Growth factor homolog zvegf4

ABSTRACT

Polypeptide growth factors, methods of making them, polynucleotides encoding them, antibodies to them, and methods of using them are disclosed. The polypeptides comprise an amino acid segment that is at least 70% identical to residues 52-179 of SEQ ID NO:2 or residues 258-370 of SEQ ID NO:2. Multimers of the polypeptides are also disclosed. The polypeptides, multimeric proteins, and polynucleotides can be used in the study and regulation of cell and tissue development, as components of cell culture media, and as diagnostic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/363,540, filed Jan. 30, 2009, which is a continuation of U.S.application Ser. No. 11/948,091, filed Nov. 30, 2007, now abandoned,which is a divisional of U.S. application Ser. No. 11/550,246, filedOct. 17, 2006, now U.S. Pat. No. 7,323,446, which is a continuation ofU.S. application Ser. No. 11/080,803, filed Mar. 15, 2005, which is acontinuation of U.S. application Ser. No. 09/876,813, filed Jun. 6,2001, now U.S. Pat. No. 6,962,802, which is a divisional of U.S.application Ser. No. 09/564,595, filed May 3, 2000, now U.S. Pat. No.6,495,668, which claims the benefit of U.S. Provisional Application Ser.No. 60/132,250 filed May 3, 1999, U.S. Provisional Application Ser. No.60/164,463, filed Nov. 10, 1999, and U.S. Provisional Application Ser.No. 60/180,169, filed Feb. 4, 2000, all of which are herein incorporatedby reference.

BACKGROUND OF THE INVENTION

In multicellular animals, cell growth, differentiation, and migrationare controlled by polypeptide growth factors. These growth factors playa role in both normal development and pathogenesis, including thedevelopment of solid tumors.

Polypeptide growth factors influence cellular events by binding tocell-surface receptors, many of which are tyrosine kinases. Bindinginitiates a chain of signalling events within the cell, which ultimatelyresults in phenotypic changes, such as cell division, proteaseproduction, and cell migration.

Growth factors can be classified into families on the basis ofstructural similarities. One such family, the PDGF (platelet derivedgrowth factor) family, is characterized by a dimeric structurestabilized by disulfide bonds. This family includes PDGF, the placentalgrowth factors (PlGFs), and the vascular endothelial growth factors(VEGFs). The individual polypeptide chains of these proteins formcharacteristic higher-order structures having a bow tie-likeconfiguration about a cystine knot, formed by disulfide bonding betweenpairs of cysteine residues. Hydrophobic interactions between loopscontribute to the dimerization of the two monomers. See, Daopin et al.,Science 257:369, 1992; Lapthorn et al., Nature 369:455, 1994. Members ofthis family are active as both homodimers and heterodimers. See, forexample, Heldin et al., EMBO J. 7:1387-1393, 1988; Cao et al., J. Biol.Chem. 271:3154-3162, 1996. The cystine knot motif and “bow tie” fold arealso characteristic of the growth factors transforming growthfactor-beta (TGF-β) and nerve growth factor (NGF), and the glycoproteinhormones. Although their amino acid sequences are quite divergent, theseproteins all contain the six conserved cysteine residues of the cystineknot.

Five vascular endothelial growth factors have been identified: VEGF,also known as vascular permeability factor (Dvorak et al., Am. J.Pathol. 146:1029-1039, 1995); VEGF-B (Olofsson et al., Proc. Natl. Acad.Sci. USA 93:2567-2581, 1996; Hayward et al., WIPO Publication WO96/27007); VEGF-C (Joukov et al., EMBO J. 15:290-298, 1996); VEGF-D(Oliviero, WO 97/12972; Achen et al., WO 98/07832), and zvegf3 (SEQ IDNO:32 and NO:33; co-pending U.S. Patent Applications Nos. 60/111,173,60/142,576, and 60/161,653). Five VEGF polypeptides (121, 145, 165, 189,and 206 amino acids) arise from alternative splicing of the VEGF mRNA.

VEGFs stimulate the development of vasculature through a process knownas angiogenesis, wherein vascular endothelial cells re-enter the cellcycle, degrade underlying basement membrane, and migrate to form newcapillary sprouts. These cells then differentiate, and mature vesselsare formed. This process of growth and differentiation is regulated by abalance of pro-angiogenic and anti-angiogenic factors. Angiogenesis iscentral to normal formation and repair of tissue, occurring in embryodevelopment and wound healing. Angiogenesis is also a factor in thedevelopment of certain diseases, including solid tumors, rheumatoidarthritis, diabetic retinopathy, macular degeneration, andatherosclerosis.

A number of proteins from vertebrates and invertebrates have beenidentified as influencing neural development. Among those molecules aremembers of the neuropilin family and the semaphorin/collapsin family.

Three receptors for VEGF have been identified: KDR/Flk-1 (Matthews etal., Proc. Natl. Acad. Sci. USA 88:9026-9030, 1991), Flt-1 (de Vries etal., Science 255:989-991, 1992), and neuropilin-1 (Soker et al., Cell92:735-745, 1998). Neuropilin-1 is also a receptor for PlGF-2 (Migdal etal., J. Biol. Chem. 273: 22272-22278, 1998).

Neuropilin-1 is a cell-surface glycoprotein that was initiallyidentified in Xenopus tadpole nervous tissues, then in chicken, mouse,and human. The primary structure of neuropilin-1 is highly conservedamong these vertebrate species. Neuropilin-1 has been demonstrated to bea receptor for various members of the semaphorin family includingsemaphorin III (Kolodkin et al., Cell 90:753-762, 1997), Sema E and SemaIV (Chen et al., Neuron 19:547-559, 1997). A variety of activities havebeen associated with the binding of neuropilin-1 to its ligands. Forexample, binding of semaphorin III to neuropilin-1 can induce neuronalgrowth cone collapse and repulsion of neurites in vitro (Kitsukawa etal., Neuron 19: 995-1005, 1997). Experiments with transgenic miceindicate the involvement of neuropilin-1 in the development of thecardiovascular system, nervous system, and limbs. See, for example,Kitsukawa et al., Development 121:4309-4318, 1995; and Takashima et al.,American Heart Association 1998 Meeting, Abstract # 3178.

Semaphorins are a large family of molecules which share the definingsemaphorin domain of approximately 500 amino acids. Dimerization isbelieved to be important for functional activity (Klostermann et al., J.Biol. Chem. 273:7326-7331, 1998). Collapsin-1, the first identifiedvertebrate member of the semaphorin family of axon guidance proteins,has also been shown to form covalent dimers, with dimerization necessaryfor collapse activity (Koppel et al., J. Biol. Chem. 273:15708-15713,1998). Semaphorin III has been associated in vitro with regulatinggrowth clone collapse and chemorepulsion of neurites. Semaphorins havebeen shown to be responsible for a variety of developmental effects,including effects on sensory afferent innervation, skeletal and cardiacdevelopment (Fehar et al., Nature 383:525-528, 1996), immunosuppressionvia inhibition of cytokines (Mangasser-Stephan et al., Biochem. Biophys.Res. Comm. 234:153-156, 1997), and promotion of B-cell aggregation anddifferentiation (Hall et al., Proc. Natl. Acad. Sci. USA 93:11780-11785,1996). CD100 has also been shown to be expressed in many T-celllymphomas and may be a marker of malignant T-cell neoplasms (Dorfman etal., Am. J. Pathol. 153:255-262, 1998). Transcription of the mousesemaphorin gene, M-semaH, correlates with metastatic ability of mousetumor cell lines (Christensen et al., Cancer Res. 58:1238-1244, 1998).

The role of growth factors, other regulatory molecules, and theirreceptors in controlling cellular processes makes them likely candidatesand targets for therapeutic intervention. Platelet-derived growthfactor, for example, has been disclosed for the treatment of periodontaldisease (U.S. Pat. No. 5,124,316), gastrointestinal ulcers (U.S. Pat.No. 5,234,908), and dermal ulcers (Robson et al., Lancet 339:23-25,1992). Inhibition of PDGF receptor activity has been shown to reduceintimal hyperplasia in injured baboon arteries (Giese et al., RestenosisSummit VIII, Poster Session #23, 1996; U.S. Pat. No. 5,620,687). PDGFhas also been shown to stimulate bone cell replication (reviewed byCanalis et al., Endocrinology and Metabolism Clinics of North America18:903-918, 1989), to stimulate the production of collagen by bone cells(Centrella et al., Endocrinology 125:13-19, 1989) and to be useful inregenerating periodontal tissue (U.S. Pat. No. 5,124,316; Lynch et al.,J. Clin. Periodontol. 16:545-548, 1989). Vascular endothelial growthfactors (VEGFs) have been shown to promote the growth of blood vesselsin ischemic limbs (Isner et al., The Lancet 348:370-374, 1996), and havebeen proposed for use as wound-healing agents, for treatment ofperiodontal disease, for promoting endothelialization in vascular graftsurgery, and for promoting collateral circulation following myocardialinfarction (WIPO Publication No. WO 95/24473; U.S. Pat. No. 5,219,739).VEGFs are also useful for promoting the growth of vascular endothelialcells in culture. A soluble VEGF receptor (soluble flt-1) has been foundto block binding of VEGF to cell-surface receptors and to inhibit thegrowth of vascular tissue in vitro (Biotechnology News 16(17):5-6,1996).

In view of the proven clinical utility of polypeptide growth factors,there is a need in the art for additional such molecules for use astherapeutic agents, diagnostic agents, and research tools and reagents.

The present invention provides such polypeptides for these and otheruses that should be apparent to those skilled in the art from theteachings herein.

DESCRIPTION OF THE INVENTION

The present invention provides an isolated polypeptide of at least 15amino acid residues comprising an epitope-bearing portion of a proteinof SEQ ID NO:2. Within certain embodiments, the polypeptide comprises asegment that is at least 70% identical to residues 52-179 of SEQ ID NO:2or residues 258-370 of SEQ ID NO:2. Within other embodiments, thepolypeptide is selected from the group consisting of residues 19-179 ofSEQ ID NO:2, residues 52-179 of SEQ ID NO:2, residues 19-257 of SEQ IDNO:2, residues 52-257 of SEQ ID NO:2, residues 19-253 of SEQ ID NO:2,residues 52-253 of SEQ ID NO:2, residues 19-370 of SEQ ID NO:2, residues52-370 of SEQ ID NO:2, residues 258-370 of SEQ ID NO:2, and residues180-370 of SEQ ID NO:2.

The invention also provides an isolated polypeptide comprising asequence of amino acids of the formula R1_(x)—R2_(y)—R3_(z), wherein R1is a polypeptide of from 100 to 130 residues in length, is at least 70%identical to residues 52-179 of SEQ ID NO:2, and comprises a sequencemotif C[KR]Y[DNE][WYF]({11,15}G[KR][WYF]C (SEQ ID NO:4) corresponding toresidues 109-131 of SEQ ID NO:2; R2 is a polypeptide at least 90%identical to residues 180-257 of SEQ ID NO:2; R3 is a polypeptide atleast 70% identical in amino acid sequence to residues 258-370 of SEQ IDNO:2 and comprises cysteine residues at positions corresponding toresidues 272, 302, 306, 318, 360, and 362 of SEQ ID NO:2, a glycineresidue at a position corresponding to residue 304 of SEQ ID NO:2, and asequence motif CX {18,33}CXGXCX{6,33}CX{20,50}CXC (SEQ ID NO:3)corresponding to residues 272-362 of SEQ ID NO:2; and each of x, y, andz is individually 0 or 1, subject to the limitations that at least oneof x and z is 1, and, if x and z are each 1, then y is 1. There are thusprovided isolated polypeptides of the above formula wherein (a) x=1, (b)z=1, and (c) x=1 and z=1. Within certain embodiments, x=1 and R1 is atleast 90% identical to residues 52-179 of SEQ ID NO:2 or residues 19-179of SEQ ID NO:2. Within related embodiments, x=1 and R1 comprisesresidues 52-179 of SEQ ID NO:2. Within other embodiments, z=1 and R3 isat least 90% identical to residues 258-370 of SEQ ID NO:2 or R3comprises residues 258-370 of SEQ ID NO:2. Within other embodiments,x=1, z=1, and R3 is at least 90% identical to residues 258-370 of SEQ IDNO:2; and x=1, z=1, R1 is at least 90% identical to residues 52-179 ofSEQ ID NO:2, and R2 is at least 90% identical to residues 180-257 of SEQID NO:2. Within additional embodiments, x=1, z=1, and the polypeptidecomprises residues 19-370 of SEQ ID NO:2 or residues 52-370 of SEQ IDNO:2. The isolated polypeptide may further comprise cysteine residues atpositions corresponding to residues 308 and 316 of SEQ ID NO:2. Withinother embodiments, the isolated polypeptide further comprises anaffinity tag. Within a related embodiment, the isolated polypeptidecomprises an immunoglobulin constant domain.

The present invention also provides an isolated protein comprising afirst polypeptide operably linked to a second polypeptide, wherein thefirst polypeptide comprises a sequence of amino acids of the formulaR1_(x)—R2_(y)—R3_(z) as disclosed above. The protein modulates cellproliferation, apoptosis, differentiation, metabolism, or migration.Within one embodiment, the protein is a heterodimer. Within relatedembodiments, the second polypeptide is selected from the groupconsisting of VEGF, VEGF-B, VEGF-C, VEGF-D, zvegf3 (SEQ ID NO:33), PlGF,PDGF-A, and PDGF-B. Within other related embodiments, x=1, z=1, and thesecond polypeptide comprises residues 46-345 of SEQ ID NO:33; x=1 andthe second polypeptide comprises residues 46-170 of SEQ ID NO:33; or z=1and the second polypeptide comprises residues 235-345 of SEQ ID NO:33.

Within another embodiment, the protein is a homodimer.

There is also provided an isolated protein produced by a methodcomprising the steps of (a) culturing a host cell containing anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a polypeptide selectedfrom the group consisting of (i) residues 52-370 of SEQ ID NO:2, (ii)residues 52-253 of SEQ ID NO:2, (iii) residues 180-370 of SEQ ID NO:2,and (iv) residues 258-370 of SEQ ID NO:2; and a transcriptionterminator, under conditions whereby the DNA segment is expressed; and(b) recovering from the cell the protein product of expression of theDNA construct.

Within another aspect of the invention there is provided an isolatedpolynucleotide of up to approximately 4.4 kb in length, wherein saidpolynucleotide encodes a polypeptide as disclosed above. Within oneembodiment of the invention, the polynucleotide is DNA.

Within a further aspect of the invention there is provided an expressionvector comprising the following operably linked elements: (a) atranscription promoter; (b) a DNA polynucleotide as disclosed above; and(c) a transcription terminator. The vector may further comprise asecretory signal sequence operably linked to the DNA polynucleotide.

Also provided by the invention is a cultured cell into which has beenintroduced an expression vector as disclosed above, wherein the cellexpresses the polypeptide encoded by the DNA polynucleotide. Thecultured cell can be used within a method of producing a polypeptide,the method comprising culturing the cell and recovering the expressedpolypeptide.

The proteins provided herein can be combined with a pharmaceuticallyacceptable vehicle to provide a pharmaceutical composition.

The invention also provides an antibody that specifically binds to anepitope of a polypeptide as disclosed above. Antibodies of the inventioninclude, inter alia, monoclonal antibodies and single chain antibodies,and may be linked to a reporter molecule.

The invention further provides a method for detecting a geneticabnormality in a patient, comprising the steps of (a) obtaining agenetic sample from a patient, (b) incubating the genetic sample with apolynucleotide comprising at least 14 contiguous nucleotides of SEQ IDNO:1 or the complement of SEQ ID NO:1, under conditions wherein thepolynucleotide will hybridize to a complementary polynucleotidesequence, to produce a first reaction product, and (c) comparing thefirst reaction product to a control reaction product, wherein adifference between the first reaction product and the control reactionproduct is indicative of a genetic abnormality in the patient.

The invention also provides a polypeptide comprising a sequence selectedfrom the group consisting of: residues 46-234 of SEQ ID NO:33 operablylinked to residues 258-370 of SEQ ID NO:2; residues 46-170 of SEQ IDNO:33 operably linked to residues 180-370 of SEQ ID NO:2; residues52-257 of SEQ ID NO:2 operably linked to residues 235-345 of SEQ IDNO:33; and residues 52-179 of SEQ ID NO:2 operably linked to residues171-345 of SEQ ID NO:33.

The invention also provides a method of activating a cell-surface PDGFreceptor, comprising exposing a cell comprising a cell-surface PDGFreceptor to a polypeptide or protein as disclosed above, whereby thepolypeptide or protein binds to and activates the receptor.

The invention also provides a method of inhibiting a PDGFreceptor-mediated cellular process, comprising exposing a cellcomprising a cell-surface PDGF receptor to a compound that inhibitsbinding of a polypeptide or protein as disclosed above to the receptor.

The invention also provides a method of stimulating the growth of bonetissue, comprising applying to bone a growth-stimulating amount of apolypeptide or protein as disclosed above.

The invention also provides a method of modulating the proliferation,differentiation, migration, or metabolism of bone cells, comprisingexposing bone cells to an effective amount of a polypeptide or proteinas disclosed above.

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention and theattached drawings.

In the accompanying drawings:

FIGS. 1A-1G are a Hopp/Woods hydrophilicity profile of the amino acidsequence shown in SEQ ID NO:2. The profile is based on a slidingsix-residue window. Buried G, S, and T residues and exposed H, Y, and Wresidues were ignored. These residues are indicated in the figure bylower case letters.

FIG. 2 is an illustration of the vector pHB12-8 for use in expressingcDNAs in transgenic animals.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), maltose binding protein(Kellerman and Ferenci, Methods Enzymol. 90:459-463, 1982; Guan et al.,Gene 67:21-30, 1987), Glu-Glu affinity tag (Grussenmeyer et al., Proc.Natl. Acad. Sci. USA 82:7952-4, 1985; see SEQ ID NO:5), substance P,Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988), streptavidinbinding peptide, thioredoxin, ubiquitin, cellulose binding protein, T7polymerase, or other antigenic epitope or binding domain. See, ingeneral, Ford et al., Protein Expression and Purification 2: 95-107,1991. DNAs encoding affinity tags and other reagents are available fromcommercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.; NewEngland Biolabs, Beverly, Mass.; and Eastman Kodak, New Haven, Conn.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

A “beta-strand-like region” is a region of a protein characterized bycertain combinations of the polypeptide backbone dihedral angles phi (φ)and psi (ω). Regions wherein φ is less than −60° and ω is greater than90° are beta-strand-like. Those skilled in the art will recognize thatthe limits of a β-strand are somewhat imprecise and may vary with thecriteria used to define them. See, for example, Richardson andRichardson in Fasman, ed., Prediction of Protein Structure and thePrinciples of Protein Conformation, Plenum Press, New York, 1989; andLesk, Protein Architecture: A Practical Approach, Oxford UniversityPress, New York, 1991.

A “complement” of a polynucleotide molecule is a polynucleotide moleculehaving a complementary base sequence and reverse orientation as comparedto a reference sequence. For example, the sequence 5′ ATGCACGGG 3′ iscomplementary to 5′ CCCGTGCAT 3′.

“Corresponding to”, when used in reference to a nucleotide or amino acidsequence, indicates the position in a second sequence that aligns withthe reference position when two sequences are optimally aligned.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolatedpolynucleotide molecules of the present invention are free of othergenes with which they are ordinarily associated, but may includenaturally occurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see, for example, Dynan and Tijan,Nature 316:774-78, 1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. Within one embodiment, the isolatedpolypeptide or protein is substantially free of other polypeptides orproteins, particularly other polypeptides or proteins of animal origin.The polypeptides or proteins may be provided in a highly purified form,i.e. greater than 95% pure or greater than 99% pure. When used in thiscontext, the term “isolated” does not exclude the presence of the samepolypeptide or protein in alternative physical forms, such as dimers oralternatively glycosylated or derivatized forms.

A “motif” is a series of amino acid positions in a protein sequence forwhich certain amino acid residues are required. A motif defines the setof possible residues at each such position.

“Operably linked” means that two or more entities are joined togethersuch that they function in concert for their intended purposes. Whenreferring to DNA segments, the phrase indicates, for example, thatcoding sequences are joined in the correct reading frame, andtranscription initiates in the promoter and proceeds through the codingsegment(s) to the terminator. When referring to polypeptides, “operablylinked” includes both covalently (e.g., by disulfide bonding) andnon-covalently (e.g., by hydrogen bonding, hydrophobic interactions, orsalt-bridge interactions) linked sequences, wherein the desiredfunction(s) of the sequences are retained.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A “secretory signal sequence” is a DNA sequence that encodes apolypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

A “segment” is a portion of a larger molecule (e.g., polynucleotide orpolypeptide) having specified attributes. For example, a DNA segmentencoding a specified polypeptide is a portion of a longer DNA molecule,such as a plasmid or plasmid fragment, that, when read from the 5′ tothe 3′ direction, encodes the sequence of amino acids of the specifiedpolypeptide.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±20%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the discovery of a novel DNAmolecule that encodes a polypeptide comprising a growth factor domainand a CUB domain. The growth factor domain is characterized by anarrangement of cysteine residues and beta strands that is characteristicof the “cystine knot” structure of the PDGF family. The CUB domain showssequence homology to CUB domains in the neuropilins (Takagi et al.,Neuron 7:295-307, 1991; Soker et al., ibid.), human bone morphogeneticprotein-1 (Wozney et al., Science 242:1528-1534, 1988), porcine seminalplasma protein and bovine acidic seminal fluid protein (Romero et al.,Nat. Struct. Biol. 4:783-788, 1997), and X. laevis tolloid-like protein(Lin et al., Dev. Growth Differ. 39:43-51, 1997). Analysis of the tissuedistribution of the mRNA corresponding to this novel DNA showed thatexpression was widespread in adult human tissues. The polypeptide hasbeen designated “zvegf4” in view of its homology to the VEGFs in thegrowth factor domain.

Structural predictions based on the zvegf4 sequence and its homology toother growth factors suggests that the polypeptide can formhomomultimers or heteromultimers that act on tissues to control organdevelopment by modulating cell proliferation, migration,differentiation, or metabolism. Experimental evidence supports thesepredictions. Zvegf4 heteromultimers may comprise a polypeptide fromanother member of the PDGF/VEGF family of proteins, including VEGF,VEGF-B, VEGF-C, VEGF-D, zvegf3, PlGF (Maglione et al., Proc. Natl. Acad.Sci. USA 88:9267-9271, 1991), PDGF-A (Murray et al., U.S. Pat. No.4,899,919; Heldin et al., U.S. Pat. No. 5,219,759), or PDGF-B (Chiu etal., Cell 37:123-129, 1984; Johnsson et al., EMBO J. 3:921-928, 1984).Members of this family of polypeptides regulate organ development andregeneration, post-developmental organ growth, and organ maintenance, aswell as tissue maintenance and repair processes. These factors are alsoinvolved in pathological processes where therapeutic treatments arerequired, including cancer, rheumatoid arthritis, diabetic retinopathy,ischemic limb disease, peripheral vascular disease, myocardial ischemia,vascular intimal hyperplasia, atherosclerosis, and hemangioma formation.To treat these pathological conditions it will often be required todevelop compounds to antagonize the members of the PDGF/VEGF family ofproteins, or their respective receptors. This may include thedevelopment of neutralizing antibodies, small molecule antagonists,modified forms of the growth factors that maintain receptor bindingactivity but lack receptor activating activity, soluble receptors(including receptor-immunoglobulin fusion proteins) or antisense orribozyme molecules to block polypeptide production.

A representative human zvegf4 polypeptide sequence is shown in SEQ IDNO:2, and a representative mouse zvegf4 polypeptide sequence is shown inSEQ ID NO:53. DNAs encoding these polypeptides are shown in SEQ ID NOS:1and 52, respectively. Analysis of the amino acid sequence shown in SEQID NO:2 indicates that residues 1 to 18 form a secretory peptide. TheCUB domain extends from residue 52 to residue 179. A propeptide-likesequence extends from residue 180 to either residue 245, residue 249 orresidue 257, and includes four potential cleavage sites at its carboxylterminus, monobasic sites at residue 245 and residue 249, a dibasic siteat residues 254-255, and a target site for furin or a furin-likeprotease at residues 254-257. Protein produced in a baculovirusexpression system showed cleavage between residues 250 and 249, as wellas longer species with amino termini at residues 19 and 35. The growthfactor domain extends from residue 258 to residue 370, and may includeadditional residues at the N-terminus (for instance, this domain mayinclude residues 250 to 370 or residues 246 to 370). Those skilled inthe art will recognize that domain boundaries are somewhat imprecise andcan be expected to vary by up to ±5 residues from the specifiedpositions. Cleavage of full-length zvegf4 with plasmin resulted inactivation of the zvegf4 polypeptide. By Western analysis, a bandmigrating at approximately the same size as the growth factor domain wasobserved. A matched, uncleaved full-length zvegf4 sample demonstrated noactivation.

Signal peptide cleavage is predicted to occur in human zvegf4 afterresidue 18 (±3 residues). Upon comparison of human and mouse zvegf4sequences, alternative signal peptide cleavage sites are predicted afterresidue 23 and/or residue 24. This analysis suggests that the zvegf4polypeptide chain may be cleaved to produce a plurality of monomericspecies, some of which are shown in Table 1. In certain host cells,cleavage after Lys-255 is expected to result in subsequent removal ofresidues 254-255, although polypeptides with a carboxyl terminus atresidue 255 may also be prepared. Cleavage after Lys-257 is expected toresult in subsequent removal of residue 257. These cleavage sites can bemodified to prevent proteolysis and thus provide for the production ofuncleaved zvegf4 polypeptides and multimers comprising them. Actualcleavage patterns are expected to vary among host cells.

TABLE 1 Monomer Residues (SEQ ID NO: 2) Cub domain 19-179 35-179 52-179CUB domain + interdomain region 19-257 35-257 52-257 19-255 35-25552-255 19-253 35-253 52-253 19-249 35-249 52-249 19-245 35-245 52-245Cub domain + interdomain region + growth 19-370 factor domain 35-37052-370 Growth factor domain 246-370  250-370  258-370  Growth factordomain + interdomain region 180-370 

Also included within the present invention are polypeptides that are atleast 70%, 80%, 90%, and 95% identical to the polypeptides disclosed inTable 1, wherein these additional polypeptides retain certaincharacteristic sequence motifs as disclosed below.

Zvegf4 polypeptides are designated herein with a subscript indicatingthe amino acid residues. For example, the CUB domain polypeptidesdisclosed in Table 1 are designated “zvegf4₁₉₋₁₇₉”, “zvegf4₃₅₋₁₇₉”, and“zvegf4₅₂₋₁₇₉”.

Higher order structure of zvegf4 polypeptides can be predicted bysequence alignment with known homologs and computer analysis usingavailable software (e.g., the Insight II® viewer and homology modelingtools; MSI, San Diego, Calif.). Analysis of SEQ ID NO:2 predicts thatthe secondary structure of the growth factor domain is dominated by thecystine knot, which ties together variable beta strand-like regions andloops into a bow tie-like structure. Sequence alignment indicates thatCys residues within the growth factor domain at positions 272, 302, 306,318, 360, and 362, and Gly 304 are highly conserved within the family.Further analysis suggests pairing (disulfide bond formation) of Cysresidues 272 and 318, 302 and 360, and 306 and 362 to form the cystineknot. This arrangement of conserved residues can be represented by theformula CX{18,33}CXGXCX{6,33}CX{20,50}CXC (SEQ ID NO:3), wherein aminoacid residues are represented by the conventional single-letter code, Xis any amino acid residue, and {y,z} indicates a region of variableresidues (X) from y to z residues in length. A consensus bow tiestructure is formed as: amino terminus to cystine knot→beta strand-likeregion 1→variable loop 1→beta strand-like region 2→cystine knot→betastrand-like region 3 variable loop 2→beta strand-like region 4→cystineknot→beta strand-like region 5→variable loop 3→beta strand-like region6→cystine knot. Variable loops 1 and 2 form one side of the bow tie,with variable loop 3 forming the other side. The structure of the zvegf4growth factor domain appears to diverge from the consensus structure ofother family members in loop 2 and beta strand-like regions 3 and 4,wherein all are abbreviated and essentially replaced by a cysteinecluster comprising residues 307 (Gly) through 317 (Thr), which includesCys residues at positions 308 and 316 of SEQ ID NO:2. The approximateboundaries of the beta strand-like regions in SEQ ID NO:2 are: region 1,residues 273-281; region 2, residues 297-301; region 5, residues319-324; region 6, residues 355-358. Loops separate regions 1 and 2, andregions 5 and 6.

The CUB domain of zvegf4 is believed to form a beta barrel structurewith nine distinct beta strand-like regions. These regions compriseresidues 54-57, 61-65, 79-84, 90-95, 97-99, 112-115, 126-130, 146-150,and 163-170 of SEQ ID NO:2. A multiple alignment of CUB domains ofXenopus laevis neuropilin precursor (Takagi et al., ibid.), human BMP-1(Wozney et al., ibid.), and X. laevis tolloid-like protein (Lin et al.,ibid.) indicates the presence of a conserved motif corresponding toresidues 109-131 of SEQ ID NO:2. This motif is represented by theformula C[KR]Y[DNE][WYF]X{11,15} G[KR][WYF]C (SEQ ID NO:4), whereinsquare brackets indicate the allowable residues at a given position andX{y,z} is as defined above.

The proteins of the present invention include proteins comprising CUBdomains homologous to the CUB domain of zvegf4. These homologous domainsare from 100 to 120 residues in length and comprise a motif of thesequence C[KR]Y[DNE][WYF]X{11,15}G[KR][WYF]C (SEQ ID NO:4) correspondingto residues 109-131 of SEQ ID NO:2. These homologous CUB domains are atleast 70% identical to residues 52-179 of SEQ ID NO:2, at least 80%identical, at least 90% identical, or at least 95% identical to residues52-179 of SEQ ID NO:2.

CUB domain-containing proteins of the present invention may furtherinclude a zvegf4 interdomain region or homolog thereof. The interdomainregion is at least 70% identical to residues 180 to 253 of SEQ ID NO:2.

As noted above, residues 254-257 of SEQ ID NO:2 are believed to providecleavage sites for furin or other proteases. However, polypeptidescomprising a C-terminal interdomain region (e.g., zvegf4₅₂₋₂₅₇) can beprepared with or without one or more of residues 254-257 at the carboxylterminus. In addition, polypeptides comprising another C-terminalinterdomain region (e.g., zvegf₄₅₂₋₂₄₅) can be prepared.

Additional proteins of the present invention comprise the zvegf4 growthfactor domain or a homolog thereof. These proteins thus comprise apolypeptide segment that is at least 70%, 80%, 90% or 95% identical toresidues 258-370 of SEQ ID NO:2, wherein the polypeptide segmentcomprises Cys residues at positions corresponding to residues 272, 302,306, 318, 360, and 362 of SEQ ID NO:2; a glycine at a positioncorresponding to residue 304 of SEQ ID NO:2; and the sequence motifCX{18,33}CXGXCX {6,33}CX{20,50}CXC (SEQ ID NO:3) corresponding toresidues 272-362 of SEQ ID NO:2.

Additional proteins comprising combinations of the CUB domain,interdomain region, and growth factor domain are shown above in Table 1.In each case, the invention also includes homologous proteins comprisinghomologous domains as disclosed above. More particularly, domains orregions in the mouse zvegf4 protein corresponding to domains or regionsin the human zvegf4 protein are included within the present invention.

Structural analysis and homology predict that zvegf4 polypeptidescomplex with a second polypeptide to form multimeric proteins. Theseproteins include homodimers and heterodimers. In the latter case, thesecond polypeptide can be a truncated or other variant zvegf4polypeptide or another polypeptide, such as a PlGF, PDGF-A, PDGF-B,VEGF, VEGF-B, VEGF-C, VEGF-D, or zvegf3 polypeptide. Among the dimericproteins within the present invention are dimers formed by non-covalentassociation (e.g., hydrophobic interactions) with a second subunit,either a second zvegf4 polypeptide or other second subunit, or bycovalent association stabilized by intermolecular disulfide bondsbetween cysteine residues of the component monomers. Within SEQ ID NO:2,the Cys residues at positions 296, 308, 316, and 364 may formintramolecular or intermolecular disulfide bonds.

The present invention thus provides a variety of multimeric proteinscomprising a zvegf4 polypeptide as disclosed above. These zvegf4polypeptides include zvegf4₁₉₋₁₇₉, zvegf4₃₅₋₁₇₉, zvegf4₅₂₋₁₇₉,zvegf4₁₉₋₂₄₅, ZVegf4₃₅₋₂₄₅, zvegf4₅₂₋₂₄₅, zvegf4₁₉₋₂₄₉, zvegf4₃₅₋₂₄₉,zvegf4₅₂₋₇₄₉, zvegf₄₁₉₋₂₅₃, zvegf4₃₅₋₂₅₃, zvegf4₅₂₋₂₅₃, zvegf4₁₉₋₂₅₅,zvegf4₃₅₋₂₅₅, zvegf4₅₂₋₂₅₅, zvegf4₁₉₋₂₅₇, zvegf4₃₅₋₂₅₇, zvegf4₅₂₋₂₅₇,zvegf4₁₉₋₃₇₀, zvegf4₃₅₋₃₇₀, zvegf4₅₂₋₃₇₀, zvegf4₂₄₆₋₃₇₀, zvegf4₂₅₀₋₃₇₀,and zvegf4₂₅₈₋₃₇₀, as well as variants and derivatives of thesepolypeptides as disclosed herein. These zvegf4 polypeptides can beprepared as homodimers or as heterodimers with corresponding regions ofrelated family members. For example, a zvegf4 CUB domain polypeptide canbe dimerized with a polypeptide comprising residues 46-170 of SEQ IDNO:33; a zvegf4 growth factor domain polypeptide can be dimerized with apolypeptide comprising residues 235-345 of SEQ ID NO:33; and a zvegf4CUB domain-interdomain-growth factor domain polypeptide can be dimerizedwith a polypeptide comprising residues 46-345 of SEQ ID NO:33.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603-616, 1986, andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 2 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as:

$\frac{{Total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {identical}\mspace{14mu} {matches}}{\begin{bmatrix}\begin{matrix}{{length}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {longer}\mspace{14mu} {sequence}\mspace{14mu} {plus}\mspace{14mu} {the}} \\{{number}\mspace{14mu} {of}\mspace{14mu} {gaps}\mspace{14mu} {introduced}\mspace{14mu} {into}\mspace{14mu} {the}\mspace{14mu} {longer}}\end{matrix} \\{{sequence}\mspace{14mu} {in}\mspace{14mu} {order}\mspace{14mu} {to}\mspace{14mu} {align}\mspace{14mu} {the}\mspace{14mu} {two}\mspace{14mu} {sequences}}\end{bmatrix}} \times 100$

TABLE 2 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

The level of identity between amino acid sequences can be determinedusing the “FASTA” similarity search algorithm of Pearson and Lipman(Proc. Natl. Acad. Sci. USA 85:2444, 1988) and Pearson (Meth. Enzymol.183:63, 1990). Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO:2) anda test sequence that have either the highest density of identities (ifthe ktup variable is 1) or pairs of identities (if ktup=2), withoutconsidering conservative amino acid substitutions, insertions, ordeletions. The ten regions with the highest density of identities arethen rescored by comparing the similarity of all paired amino acidsusing an amino acid substitution matrix, and the ends of the regions are“trimmed” to include only those residues that contribute to the highestscore. If there are several regions with scores greater than the“cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, 1990 (ibid.).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom four to six.

Within certain embodiments of the invention amino acid substitutions ascompared with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:53 areconservative substitutions. The BLOSUM62 matrix (Table 2) is an aminoacid substitution matrix derived from about 2,000 local multiplealignments of protein sequence segments, representing highly conservedregions of more than 500 groups of related proteins (Henikoff andHenikoff, ibid.). Thus, the BLOSUM62 substitution frequencies can beused to define conservative amino acid substitutions that may beintroduced into the amino acid sequences of the present invention. Asused herein, the term “conservative amino acid substitution” refers to asubstitution represented by a BLOSUM62 value of greater than −1. Forexample, an amino acid substitution is conservative if the substitutionis characterized by a BLOSUM62 value of 0, 1, 2, or 3. More conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 1 (e.g., 1, 2 or 3), while still more conservative amino acidsubstitutions are characterized by a BLOSUM62 value of at least 2 (e.g.,2 or 3).

Polypeptides of the present invention can be prepared with one or moreamino acid substitutions, deletions or additions as compared to SEQ IDNO:2 or SEQ ID NO:53. These changes can be of a minor nature, that isconservative amino acid substitutions and other changes that do notsignificantly affect the folding or activity of the protein orpolypeptide, and include amino- or carboxyl-terminal extensions, such asan amino-terminal methionine residue, an amino or carboxyl-terminalcysteine residue to facilitate subsequent linking to maleimide-activatedkeyhole limpet hemocyanin, a small linker peptide of up to about 20-25residues, or an affinity tag as disclosed above. Two or more affinitytags may be used in combination. Polypeptides comprising affinity tagscan further comprise a polypeptide linker and/or a proteolytic cleavagesite between the zvegf4 polypeptide and the affinity tag. Exemplarycleavage sites include, without limitation, thrombin cleavage sites andfactor Xa cleavage sites.

The present invention further provides a variety of other polypeptidefusions and related multimeric proteins comprising one or morepolypeptide fusions. For example, a zvegf4 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584. Exemplary dimerizing proteins in this regardinclude immunoglobulin constant region domains. Dimerization can also bestabilized by fusing a zvegf4 polypeptide to a leucine zipper sequence(Riley et al., Protein Eng. 9:223-230, 1996; Mohamed et al., J. SteroidBiochem. Mol. Biol. 51:241-250, 1994). Immunoglobulin-zvegf4 polypeptidefusions and leucine zipper fusions can be expressed in geneticallyengineered cells to produce a variety of multimeric zvegf4 analogs.Auxiliary domains can be fused to zvegf4 polypeptides to target them tospecific cells, tissues, or macromolecules (e.g., collagen). Forexample, a zvegf4 polypeptide or protein can be targeted to apredetermined cell type by fusing a zvegf4 polypeptide to a ligand thatspecifically binds to a receptor on the surface of the target cell. Inthis way, polypeptides and proteins can be targeted for therapeutic ordiagnostic purposes. A zvegf4 polypeptide can be fused to two or moremoieties, such as an affinity tag for purification and a targetingdomain. Polypeptide fusions can also comprise one or more cleavagesites, particularly between domains. See, Tuan et al., Connective TissueResearch 34:1-9, 1996.

Zvegf4 polypeptide fusions will generally contain not more than about1,500 amino acid residues, often not more than about 1,200 residues,more often not more than about 1,000 residues, and will in many cases beconsiderably smaller. For example, a zvegf4 polypeptide of 352 residues(residues 19-370 of SEQ ID NO:2) can be fused to E. coli β-galactosidase(1,021 residues; see Casadaban et al., J. Bacteriol. 143:971-980, 1980),a 10-residue spacer, and a 4-residue factor Xa cleavage site to yield apolypeptide of 1,387 residues. In a second example, residues 250-370 ofSEQ ID NO:2 can be fused to maltose binding protein (approximately 370residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag.

A polypeptide comprising the zvegf4 growth factor domain (e.g.,zvegf4₂₅₈₋₃₇₀ or zvegf4₁₈₀₋₃₇₀) may be fused to a non-zvegf4 CUB domain.Within a related embodiment of the invention, a zvegf4 polypeptidecomprising zvegf4 growth factor and CUB domains is fused to a non-zvegf4CUB domain, such as a CUB-domain-comprising neuropilin polypeptide.

The present invention further provides polypeptide fusions comprisingthe zvegf4 CUB domain (e.g., zvegf4₅₂₋₁₇₉). The CUB domain, with itshomology to neuropilin-1, may be used to target zvegf4 or other proteinscontaining it to cells having cell-surface semaphorins, includingendothelial cells, neuronal cells, lymphocytes, and tumor cells. Thezvegf4 CUB domain can thus be joined to other moieties, includingpolypeptides (e.g., other growth factors, antibodies, and enzymes) andnon-peptidic moieties (e.g., radionuclides, contrast agents, and thelike), to target them to cells expressing cell-surface semaphorins. Thecleavage sites between the CUB and growth factor domains of zvegf4 mayallow for proteolytic release of the growth factor domain or othermoiety through existing local proteases within tissues, or by proteasesadded from exogenous sources. The release of the targeted moiety mayprovide more localized biological effects.

The polypeptide fusions of the present invention further include fusionsbetween zvegf4 and zvegf3, wherein a domain of zvegf4 is replaced withthe corresponding domain of zvegf3 or a variant thereof. Arepresentative human zvegf3 polypeptide sequence is shown in SEQ IDNO:33, and a representative mouse sequence is shown in SEQ ID NO:35.Within SEQ ID NO:33, the CUB domain comprises residues 46-170, theinterdomain region comprises residues 171-234, and the growth factordomain comprises residues 235-345 (all ±5 residues). A secretory peptideis predicted to be cleaved from the polypeptide after residue 14 (±3residues). Cleavage sites are predicted at residue 249, residues254-255, and residues 254-257. Domain boundaries in mouse zvegf3 andother orthologous sequences can be determined readily by those ofordinary skill in the art by alignment with the human sequence disclosedherein. Of particular interest are fusions in which the zvegf3 CUBdomain is combined with the zvegf4 growth factor domain, and fusions inwhich the zvegf4 CUB domain is combined with the zvegf3 growth factordomain. Within these polypeptide fusions the interdomain region may bederived from either zvegf3 or zvegf4. Polypeptide fusions comprisingzvegf3 and zvegf4 sequences include both full-length and truncatedsequences, for example sequences analogous to those disclosed in Table1, above.

Proteins comprising the wild-type zvegf4 CUB domain and variants thereofmay be used to modulate activities mediated by cell-surface semaphorins.While not wishing to be bound by theory, zvegf4 may bind to semaphorinsvia its CUB domain. The observation that semaphorin III is involved invascular development suggests that members of the vascular growth factorfamily of proteins may also be involved, especially due to theco-binding activity of VEGF and semaphorin III to neuropilin-1. Zvegf4may thus be used to design agonists and antagonist ofneuropilin-semaphorin interactions. For example, the zvegf4 sequencedisclosed herein provides a starting point for the design of moleculesthat antagonize semaphorin-stimulated activities, including neuritegrowth, cardiovascular development, cartilage and limb development, andT and B-cell function. Additional applications include intervention invarious pathologies, including rheumatoid arthritis, various forms ofcancer, autoimmune disease, inflammation, retinopathies, hemangiomas,ischemic events within tissues including the heart, kidney andperipheral arteries, neuropathies, acute nerve damage, and diseases ofthe central and peripheral nervous systems, including stroke.

The isolated CUB domain of either mouse or human zvegf4 (and multimersthereof) may also be useful to block binding of other zvegf4 molecules(e.g., full-length polypeptide, isolated growth factor domain, ormultimers thereof) to cell-surface molecules and/or extracellularbinding sites by itself binding to such molecules or sites. In addition,the isolated CUB domain of either mouse or human zvegf4 may be useful toblock zvegf4 binding, and/or more generally vascular endothelial growthfactor binding, to neuropilin-1 (see M. L. Gagnon et al., Proc. Natl.Acad. Sci. USA 97:2573-78, 2000). Further, the second major loop ofzvegf4 (residues 308-316) may represent the receptor-binding loop ofzvegf4 (see, for instance, WO 99/13329; WO 98/10795; and W. J.LaRochelle et al., J. Biol. Chem. 267:17074-77, 1992), and thus may beuseful as an antagonist of zvegf4 activity. Within this peptide (zvegf4residues 308-316), Cys308 and Cys316 may or may not be disulfide bonded.Also, dimers of this peptide may be constructed such that residue Cys308is disulfide bonded to either Cys308 or Cys316 of the homodimer partnerpeptide.

Amino acid sequence changes are made in zvegf4 polypeptides so as tominimize disruption of higher order structure essential to biologicalactivity. As noted above, conservative amino acid changes are generallyless likely to negate activity than are non-conservative changes.Changes in amino acid residues will be made so as not to disrupt thecystine knot and “bow tie” arrangement of loops in the growth factordomain that is characteristic of the protein family. Conserved motifswill also be maintained. The effects of amino acid sequence changes canbe predicted by computer modeling as disclosed above or determined byanalysis of crystal structure (see, e.g., Lapthorn et al., ibid.). Ahydrophilicity profile of SEQ ID NO:2 is shown in FIGS. 1A-1H. Thoseskilled in the art will recognize that this hydrophilicity will be takeninto account when designing alterations in the amino acid sequence of azvegf4 polypeptide, so as not to disrupt the overall profile. Additionalguidance in selecting amino acid substitutions is provided by acomparison of the mouse (SEQ ID NO:53) and human (SEQ ID NO:2) zvegf4sequences. The amino acid sequence is highly conserved between mouse andhuman zvegf4s, with an overall amino acid sequence identity of 85.1%.

The polypeptides of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are knownin the art for incorporating non-naturally occurring amino acid residuesinto proteins. For example, an in vitro system can be employed whereinnonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is carried out in a cell-freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science259:806-809, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145-10149, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-19998, 1996). Within a third method, E. coli cells arecultured in the absence of a natural amino acid that is to be replaced(e.g., phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad.Sci. USA 88:4498-4502, 1991). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity of otherproperties to identify amino acid residues that are critical to theactivity of the molecule.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

Variants of the disclosed zvegf4 DNA and polypeptide sequences can begenerated through DNA shuffling as disclosed by Stemmer, Nature370:389-391, 1994 and Stemmer, Proc. Natl. Acad. Sci. USA91:10747-10751, 1994. Briefly, variant genes are generated by in vitrohomologous recombination by random fragmentation of a parent genefollowed by reassembly using PCR, resulting in randomly introduced pointmutations. This technique can be modified by using a family of parentgenes, such as allelic variants or genes from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

Mutagenesis methods as disclosed above can be combined with high volumeor high-throughput screening methods to detect biological activity ofzvegf4 variant polypeptides, in particular biological activity inmodulating cell proliferation or cell differentiation. For example,mitogenesis assays that measure dye incorporation or ³H-thymidineincorporation can be carried out on large numbers of samples, as cancell-based assays that detect expression of a reporter gene (e.g., aluciferase gene). Mutagenesis of the CUB domain can be used to modulateits binding to members of the semaphorin family, including enhancing orinhibiting binding to selected family members. A modified spectrum ofbinding activity may be desirable for optimizing therapeutic and/ordiagnostic utility of proteins comprising a zvegf4 CUB domain. Directbinding utilizing labeled CUB protein can be used to monitor changes inCUB domain binding activity to selected semaphorin family members.Semaphorins of interest in this regard include isolated proteins,proteins present in cell membranes, and proteins present oncell-surfaces. The CUB domain can be labeled by a variety of methodsincluding radiolabeling with isotopes, such as ¹²⁵I, conjugation toenzymes such as alkaline phosphatase or horseradish peroxidase,conjugation with biotin, and conjugation with various fluorescentmarkers including FITC. These and other assays are disclosed in moredetail below. Mutagenized DNA molecules that encode active zvegf4polypeptides can be recovered from the host cells and rapidly sequencedusing modern equipment. These methods allow the rapid determination ofthe importance of individual amino acid residues in a polypeptide ofinterest, and can be applied to polypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptides that are homologous tothe zvegf4 polypeptides disclosed above in Table 1 and retain thebiological properties of the wild-type protein. Such polypeptides canalso include additional polypeptide segments as generally disclosedabove.

The present invention also provides polynucleotide molecules, includingDNA and RNA molecules, that encode the zvegf4 polypeptides disclosedabove. The polynucleotides of the present invention include the sensestrand; the anti-sense strand; and the DNA as double-stranded, havingboth the sense and anti-sense strands annealed together by hydrogenbonds. A representative DNA sequence encoding human zvegf4 polypeptidesis set forth in SEQ ID NO:1, and a representative DNA sequence encodingmouse zvegf4 polypeptides is set forth in SEQ ID NO:52. Additional DNAsequences encoding zvegf4 polypeptides can be readily generated by thoseof ordinary skill in the art based on the genetic code. Counterpart RNAsequences can be generated by substitution of U for T.

Those skilled in the art will readily recognize that, in view of thedegeneracy of the genetic code, considerable sequence variation ispossible among polynucleotide molecules encoding zvegf4 polypeptides.SEQ ID NO:6 is a degenerate DNA sequence that encompasses all DNAs thatencode the zvegf4 polypeptide of SEQ ID NO: 2. Those skilled in the artwill recognize that the degenerate sequence of SEQ ID NO:6 also providesall RNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus,zvegf4 polypeptide-encoding polynucleotides comprising nucleotides1-1110, 1-537, 55-537, 103-537, 154-537, 55-771, 103-771, 154-771,55-765, 103-765, 154-765, 55-759, 103-759, 154-759, 55-747, 103-747,154-747, 55-735, 103-735, 154-735, 55-1110, 103-1110, 154-1110,772-1110, 748-1110, 736-1110, and 538-1110 of SEQ ID NO:6 and their RNAequivalents are contemplated by the present invention. Table 3 setsforth the one-letter codes used within SEQ ID NO:6 to denote degeneratenucleotide positions. “Resolutions” are the nucleotides denoted by acode letter. “Complement” indicates the code for the complementarynucleotide(s). For example, the code Y denotes either C or T, and itscomplement R denotes A or G, A being complementary to T, and G beingcomplementary to C.

TABLE 3 Nucleotide Resolutions Complement Resolutions A A T T C C G G GG C C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G SC|G W A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|TH A|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:6, encompassing all possiblecodons for a given amino acid, are set forth in Table 4, below.

TABLE 4 One- Amino Letter Degenerate Acid Code Codons Codon Cys C TGC,TGT TGY Ser S AGC, AGT, TCA, TCC, TCG, TCT WSN Thr T ACA, ACC, ACG, ACTCAN Pro P CCA, CCC, CCG, CCT CCN Ala A GCA, GCC, GCG, GCT GCN Gly G GGA,GGC, GGG, GGT GGN Asn N AAC, AAT AAY Asp D GAC, GAT GAY Glu E GAA, GAGGAR Gln Q CAA, CAG CAR His H CAC, CAT CAY Arg R AGA, AGG, CGA, CGC, CGG,CGT MGN Lys K AAA, AAG AAR Met M ATG ATG Ile I ATA, ATC, ATT ATH Leu LCTA, CTC, CTG, CTT, TTA, TTG YTN Val V GTA, GTC, GTG, GTT GTN Phe F TTC,TTT TTY Tyr Y TAC, TAT TAY Trp W TGG TGG Ter . TAA, TAG, TGA TRR Asn|AspB RAY Glu|Gln Z SAR Any X NNN Gap — —

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequences may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO: 2 and of SEQ ID NO:53. Variant sequences can bereadily tested for functionality as described herein.

Within certain embodiments of the invention the isolated polynucleotideswill hybridize to similar sized regions of SEQ ID NO:1 or SEQ ID NO:52,or a sequence complementary thereto, under stringent conditions. Ingeneral, stringent conditions are selected to be about 5° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Typical stringent conditions are those in whichthe salt concentration is up to about 0.03 M at pH 7 and the temperatureis at least about 60° C.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. Complementary DNA (cDNA) clones are prepared fromRNA that is isolated from a tissue or cell that produces large amountsof zvegf4 RNA. Such tissues and cells are identified by Northernblotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and includeheart, pancreas, stomach, and adrenal gland. Total RNA can be preparedusing guanidine HCl extraction followed by isolation by centrifugationin a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly(A)⁺ RNA is prepared from total RNA using the method of Aviv and Leder(Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972). Complementary DNA(cDNA) is prepared from poly(A)⁺ RNA using known methods. In thealternative, genomic DNA can be isolated. For some applications (e.g.,expression in transgenic animals) it may be advantageous to use agenomic clone, or to modify a cDNA clone to include at least one genomicintron. Methods for identifying and isolating cDNA and genomic clonesare well known and within the level of ordinary skill in the art, andinclude the use of the sequence disclosed herein, or parts thereof, forprobing or priming a library. Polynucleotides encoding zvegf4polypeptides are identified and isolated by, for example, hybridizationor polymerase chain reaction (“PCR”, Mullis, U.S. Pat. No. 4,683,202).Expression libraries can be probed with antibodies to zvegf4, receptorfragments, or other specific binding partners.

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NOS:1 and 2 represent a single allele of human zvegf4, and thatthe sequences disclosed in SEQ ID NOS:52 and 53 represent a singleallele of mouse zvegf4. Allelic variants of these sequences can becloned by probing cDNA or genomic libraries from different individualsaccording to standard procedures. Alternatively spliced forms of zvegf4are also expected to exist.

The zvegf4 polynucleotide sequence disclosed herein can be used toisolate polynucleotides encoding other zvegf4 proteins. Such otherpolynucleotides include allelic variants, alternatively spliced cDNAsand counterpart polynucleotides from other species (orthologs). Theseorthologous polynucleotides can be used, inter alia, to prepare therespective orthologous proteins. Other species of interest include, butare not limited to, mammalian, avian, amphibian, reptile, fish, insectand other vertebrate and invertebrate species. Of particular interestare zvegf4 polynucleotides and proteins from other mammalian species,including non-human primate, murine, porcine, ovine, bovine, canine,feline, and equine polynucleotides and proteins. Orthologs of humanzvegf4 can be cloned using information and compositions provided by thepresent invention in combination with conventional cloning techniques.For example, a cDNA can be cloned using mRNA obtained from a tissue orcell type that expresses zvegf4 as disclosed herein. Suitable sources ofmRNA can be identified by probing Northern blots with probes designedfrom the sequences disclosed herein. A library is then prepared frommRNA of a positive tissue or cell line. A zvegf4-encoding cDNA can thenbe isolated by a variety of methods, such as by probing with a completeor partial human cDNA or with one or more sets of degenerate probesbased on the disclosed sequences. Hybridization will generally be doneunder low stringency conditions, wherein washing is carried out in 1×SSCwith an initial wash at 40° C. and with subsequent washes at 5° C.higher intervals until background is suitably reduced. A cDNA can alsobe cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Pat.No. 4,683,202), using primers designed from the representative humanzvegf4 sequence disclosed herein. Within an additional method, the cDNAlibrary can be used to transform or transfect host cells, and expressionof the cDNA of interest can be detected with an antibody to zvegf4polypeptide. Similar techniques can also be applied to the isolation ofgenomic clones.

For any zvegf4 polypeptide, including variants and fusion proteins, oneof ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 3 and 4, above.

Conserved regions of zvegf4, identified by alignment with sequences ofother family members, can be used to identify related polynucleotidesand proteins. For instance, reverse transcription-polymerase chainreaction (RT-PCR) and other techniques known in the art can be used toamplify sequences encoding the conserved motifs present in zvegf4 fromRNA obtained from a variety of tissue sources. In particular, highlydegenerate primers as shown below in Table 5 (designed from an alignmentof zvegf4 with PDGF A and B chains, VEGF, VEGF-B, VEGF-C, VEGF-D, andzvegf3) are useful for cloning polynucleotides encoding homologousgrowth factor domains. Primers shown in Table 6, designed from analignment of zvegf4 with X. laevis neuropilin precursor, human BMP-1,human zvegf3, and X. laevis tolloid-like protein, are useful for cloningpolynucleotides encoding CUB domains. The primers of Tables 5 and 6 canthus be used to obtain additional polynucleotides encoding homologs ofthe zvegf4 sequence of SEQ ID NO:1 and NO:2.

TABLE 5 zvegf4 residues 301-305 degenerate: MGN TGY GGN GGN AAY TG(SEQ ID NO: 7) consensus: MGN TGY DSN GGN WRY TG (SEQ ID NO: 8)complement: CAR YWN CCN SHR CAN CK (SEQ ID NO: 9) zveg4 redidues 292-297degenerate: TTY TTY CCN MGN TGY YT (SEQ ID NO: 10) consensus:NTN DDN CCN NSN TGY BT (SEQ ID NO: 11) complement:AVR CAN SNN GGN HHN AN (SEQ ID NO: 12) zveg4 residues 357-362 degenerateCAY GAR MGN TGY GAY TG (SEQ ID NO: 13) consensus: CAY NNN NVN TGY VVN TG(SEQ ID NO: 14) complement: CAN BBR CAN BNN NNR TG (SEQ ID NO: 15)zvegf4 residues 250-255 degenerate: TGY ACN CCN MGN AAY TA(SEQ ID NO: 16) consensus: TGY HNN MCN MKN RMN DH (SEQ ID NO: 17)complement: DHN KYN MKN GKN NDR CA (SEQ ID NO: 18)

TABLE 6 zvegf4 residues 110-115 consensus: N TAY GAY TWY GTN GAR GT(SEQ ID NO: 19) complement: N ATR CTR AWR CAN CTY CA (SEQ ID NO: 20)zvegf4 residues 68-73 consensus: GN TDB CCN MAN DVN TAY C(SEQ ID NO: 21) complement: CN AHV GGN KTN HBN ATR G (SEQ ID NO: 22)zvegf4 residues 126-131 consensus: TN HDN GGN MRN TDB TGY G(SEQ ID NO: 23) complement: AN DHN CCN KYN AHV ACR C (SEQ ID NO: 24)

Zvegf4 polynucleotide sequences disclosed herein can also be used asprobes or primers to clone 5′ non-coding regions of a zvegf4 gene,including promoter sequences. A human zvegf4 genomic fragment,comprising 5′ non-coding and coding sequences, is shown in SEQ ID NO:36.These flanking sequences can be used to direct the expression of zvegf4and other recombinant proteins. In addition, 5′ flanking sequences canbe used as targeting sites for regulatory constructs to activate orincrease expression of endogenous zvegf4 genes as disclosed by Treco etal., U.S. Pat. No. 5,641,670. A human zvegf4 genomic sequence comprising5′ non-coding sequence and approximately 100 nucleotides of codingsequence is shown in SEQ ID NO:36.

The polynucleotides of the present invention can also be prepared byautomated synthesis. The production of short, double-stranded segments(60 to 80 bp) is technically straightforward and can be accomplished bysynthesizing the complementary strands and then annealing them. Longersegments (typically >300 bp) are assembled in modular form fromsingle-stranded fragments that are from 20 to 100 nucleotides in length.Automated synthesis of polynucleotides is within the level of ordinaryskill in the art, and suitable equipment and reagents are available fromcommercial suppliers. See, in general, Glick and Pasternak, MolecularBiotechnology, Principles & Applications of Recombinant DNA, ASM Press,Washington, D.C., 1994; Itakura et al., Ann. Rev. Biochem. 53: 323-56,1984; and Climie et al., Proc. Natl. Acad. Sci. USA 87:633-7, 1990.

The polypeptides of the present invention, including full-lengthpolypeptides, biologically active fragments, and fusion polypeptides canbe produced in genetically engineered host cells according toconventional techniques. Suitable host cells are those cell types thatcan be transformed or transfected with exogenous DNA and grown inculture, and include bacteria, fungal cells, and cultured highereukaryotic cells, including cultured cells of multicellular organisms.Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al.,eds., Current Protocols in Molecular Biology, Green and Wiley and Sons,NY, 1993.

In general, a DNA sequence encoding a zvegf4 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors, and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a zvegf4 polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of zvegf4, or may be derivedfrom another secreted protein (e.g., t-PA; see, U.S. Pat. No. 5,641,655)or synthesized de novo. The secretory signal sequence is operably linkedto the zvegf4 DNA sequence, i.e., the two sequences are joined in thecorrect reading frame and positioned to direct the newly synthesizedpolypeptide into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe polypeptide of interest, although certain signal sequences may bepositioned elsewhere in the DNA sequence of interest (see, e.g., Welchet al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.5,143,830).

Expression of zvegf4 polypeptides via a host cell secretory pathway isexpected to result in the production of multimeric proteins. As notedabove, such multimers include both homomultimers and heteromultimers,the latter including proteins comprising only zvegf4 polypeptides andproteins including zvegf4 and heterologous polypeptides. For example, aheteromultimer comprising a zvegf4 polypeptide and a polypeptide from arelated family member (e.g., VEGF, VEGF-B, VEGF-C, VEGF-D, zvegf3, PlGF,PDGF-A, or PDGF-B) can be produced by co-expression of the twopolypeptides in a host cell. Sequences encoding these other familymembers are known. See, for example, Dvorak et al, ibid.; Olofsson etal, ibid.; Hayward et al., ibid.; Joukov et al., ibid.; Oliviero et al.,ibid.; Achen et al., ibid.; Maglione et al., ibid.; Heldin et al., U.S.Pat. No. 5,219,759; and Johnsson et al., ibid. If a mixture of proteinsresults from expression, individual species are isolated by conventionalmethods. Monomers, dimers, and higher order multimers are separated by,for example, size exclusion chromatography. Heteromultimers can beseparated from homomultimers by conventional chromatography or byimmunoaffinity chromatography using antibodies specific for individualdimers or by sequential immunoaffinity steps using antibodies specificfor individual component polypeptides. See, in general, U.S. Pat. No.5,094,941.

Cultured mammalian cells are suitable hosts for use within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993). The production of recombinant polypeptides in cultured mammaliancells is disclosed by, for example, Levinson et al., U.S. Pat. No.4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S.Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134. Suitablecultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7(ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72,1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) celllines. Additional suitable cell lines are known in the art and availablefrom public depositories such as the American Type Culture Collection,Manassas, Va. Strong transcription promoters can be used, such aspromoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No.4,956,288. Other suitable promoters include those from metallothioneingenes (U.S. Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus majorlate promoter. Expression vectors for use in mammalian cells includepZP-1 and pZP-9, which have been deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. USA underaccession numbers 98669 and 98668, respectively.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Anexemplary selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.An exemplary amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used.

Other higher eukaryotic cells can also be used as hosts, includinginsect cells, plant cells and avian cells. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.Transformation of insect cells and production of foreign polypeptidestherein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222 and WIPOpublication WO 94/06463.

Insect cells can be infected with recombinant baculovirus, commonlyderived from Autographa californica nuclear polyhedrosis virus (AcNPV).See, King and Possee, The Baculovirus Expression System: A LaboratoryGuide, London, Chapman & Hall; O'Reilly et al., Baculovirus ExpressionVectors: A Laboratory Manual, New York, Oxford University Press., 1994;and Richardson, Ed., Baculovirus Expression Protocols. Methods inMolecular Biology, Humana Press, Totowa, N.J., 1995. Recombinantbaculovirus can also be produced through the use of a transposon-basedsystem described by Luckow et al. (J. Virol. 67:4566-4579, 1993). Thissystem, which utilizes transfer vectors, is commercially available inkit form (Bac-to-Bac™ kit; Life Technologies, Rockville, Md.). Thetransfer vector (e.g., pFastBac1™; Life Technologies) contains a Tn7transposon to move the DNA encoding the protein of interest into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990;Bonning et al., J. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk andRapoport, J. Biol. Chem. 270:1543-1549, 1995. In addition, transfervectors can include an in-frame fusion with DNA encoding a polypeptideextension or affinity tag as disclosed above. Using techniques known inthe art, a transfer vector containing a zvegf4-encoding sequence istransformed into E. coli host cells, and the cells are screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is isolated, using common techniques, and used to transfectSpodoptera frugiperda cells, such as Sf9 cells. Recombinant virus thatexpresses zvegf4 protein is subsequently produced. Recombinant viralstocks are made by methods commonly used the art.

For protein production, the recombinant virus is used to infect hostcells, typically a cell line derived from the fall armyworm, Spodopterafrugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., HighFive™ cells; Invitrogen, Carlsbad, Calif.). See, in general, Glick andPasternak, Molecular Biotechnology: Principles and Applications ofRecombinant DNA, ASM Press, Washington, D.C., 1994. See also, U.S. Pat.No. 5,300,435. Serum-free media are used to grow and maintain the cells.Suitable media formulations are known in the art and can be obtainedfrom commercial suppliers. The cells are grown up from an inoculationdensity of approximately 2-5×10⁵ cells to a density of 1-2×10⁶ cells, atwhich time a recombinant viral stock is added at a multiplicity ofinfection (MOI) of 0.1 to 10, more typically near 3. Procedures used aregenerally described in available laboratory manuals (e.g., King andPossee, ibid.; O'Reilly et al., ibid.; Richardson, ibid.).

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). An exemplary vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactic, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986; Cregg, U.S. Pat. No. 4,882,279; andRaymond et al., Yeast 14, 11-23, 1998. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533. Production ofrecombinant proteins in Pichia methanolica is disclosed in U.S. Pat.Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a zvegf4polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the alternative, the protein maybe recovered from the cytoplasm in soluble form and isolated without theuse of denaturants. The protein is recovered from the cell as an aqueousextract in, for example, phosphate buffered saline. To capture theprotein of interest, the extract is applied directly to achromatographic medium, such as an immobilized antibody orheparin-Sepharose column. Secreted polypeptides can be recovered fromthe periplasmic space in a soluble and functional form by disrupting thecells (by, for example, sonication or osmotic shock) to release thecontents of the periplasmic space and recovering the protein, therebyobviating the need for denaturation and refolding.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells, for example, are cultured in a medium comprising adequate sourcesof carbon, nitrogen and trace nutrients at a temperature of about 25° C.to 35° C. Liquid cultures are provided with sufficient aeration byconventional means, such as shaking of small flasks or sparging offermentors.

Zvegf4 polypeptides or fragments thereof can also be prepared throughchemical synthesis according to methods known in the art, includingexclusive solid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. See, for example,Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid PhasePeptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, Ill.,1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford,1989.

Covalent, multimeric complexes can also be made by isolating the desiredcomponent polypeptides and combining them in vitro. Covalent complexesthat can be prepared in this manner include homodimers of zvegf4polypeptides, heterodimers of two different zvegf4 polypeptides, andheterodimers of a zvegf4 polypeptide and a polypeptide from anotherfamily member of the VEGF/PDGF family of proteins. The two polypeptidesare mixed together under denaturing and reducing conditions, followed byrenaturation of the proteins by removal of the denaturants. Removal canbe done by, for example, dialysis or size exclusion chromatography toprovide for buffer exchange. When combining two different polypeptides,the resulting renaturated proteins may form homodimers of the individualcomponents as well as heterodimers of the two polypeptide components.See, Cao et al., J. Biol. Chem. 271:3154-3162, 1996.

Non-covalent complexes comprising a zvegf4 polypeptide can be preparedby incubating a zvegf4 polypeptide and a second polypeptide (e.g., azvegf4 polypeptide or another peptide of the PDGF/VEGF family) atnear-physiological pH. In a typical reaction, polypeptides at aconcentration of about 0.1-0.5 μg/μl are incubated at pH≈7.4 in a weakbuffer (e.g., 0.01 M phosphate or acetate buffer); sodium chloride maybe included at a concentration of about 0.1 M. At 37° C. the reaction isessentially complete with 4-24 hours. See, for example, Weintraub etal., Endocrinology 101:225-235, 1997.

Depending upon the intended use, the polypeptides and proteins of thepresent invention can be purified to ≧80% purity, ≧90% purity, ≧95%purity, or to a pharmaceutically pure state, that is greater than 99.9%pure with respect to contaminating macromolecules, particularly otherproteins and nucleic acids, and free of infectious and pyrogenic agents.

Zvegf4 proteins (including chimeric polypeptides and polypeptidemultimers) can be purified using fractionation and/or conventionalpurification methods and media, such as by a combination ofchromatographic techniques. See, in general, Affinity Chromatography:Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden,1988; and Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York, 1994. Proteins comprising a polyhistidineaffinity tag (typically about 6 histidine residues) are purified byaffinity chromatography on a nickel or cobalt chelate resin. See, forexample, Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteinscomprising a Glu-Glu tag can be purified by immunoaffinitychromatography according to conventional procedures. See, for example,Grussenmeyer et al., ibid. Maltose binding protein fusions are purifiedon an amylose column according to methods known in the art.

Using methods known in the art, zvegf4 proteins can be prepared asmonomers or multimers, glycosylated or non-glycosylated, pegylated ornon-pegylated, and may or may not include an initial methionine aminoacid residue.

The invention further provides polypeptides that comprise anepitope-bearing portion of a protein as shown in SEQ ID NO:2. An“epitope” is a region of a protein to which an antibody can bind. See,for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002,1984. Epitopes can be linear or conformational, the latter beingcomposed of discontinuous regions of the protein that form an epitopeupon folding of the protein. Linear epitopes are generally at least 6amino acid residues in length. Relatively short synthetic peptides thatmimic part of a protein sequence are routinely capable of eliciting anantiserum that reacts with the partially mimicked protein. See,Sutcliffe et al., Science 219:660-666, 1983. Antibodies that recognizeshort, linear epitopes are particularly useful in analytic anddiagnostic applications that employ denatured protein, such as Westernblotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979).Anti-peptide antibodies are not conformation-dependent and can be usedto detect proteins in fragmented or otherwise altered forms (Niman etal., Proc. Natl. Acad. Sci. USA 82:7924-7928, 1985), such as might occurin body fluids or cell culture media. Antibodies to short peptides mayalso recognize proteins in native conformation and will thus be usefulfor monitoring protein expression and protein isolation, and indetecting zvegf4 proteins in solution, such as by ELISA or inimmunoprecipitation studies.

Antigenic, epitope-bearing polypeptides of the present invention areuseful for raising antibodies, including monoclonal antibodies, thatspecifically bind to a zvegf4 protein. Antigenic, epitope-bearingpolypeptides contain a sequence of at least six, within otherembodiments at least nine, within other embodiments from 15 to about 30contiguous amino acid residues of a zvegf4 protein (e.g., SEQ ID NO:2).Polypeptides comprising a larger portion of a zvegf4 protein, i.e., from30 to 50 or 100 residues or up to the entire sequence are included. Itis preferred that the amino acid sequence of the epitope-bearingpolypeptide is selected to provide substantial solubility in aqueoussolvents, that is the sequence includes relatively hydrophilic residues,and hydrophobic residues are substantially avoided. Such regions of SEQID NO:2 include, for example, residues 39-44, 252-257, 102-107, 264-269,and 339-344. Exemplary longer peptide immunogens include peptidescomprising residues (i) 131-148, (ii) 230-253, or (iii) 333-355 of SEQID NO:2. Peptide (ii) can be prepared with an additional C-terminal cysresidue and peptide (iii) with an additional N-terminal cys residue tofacilitate coupling.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Monoclonal antibodies can also beproduced in mice that have been genetically altered to produceantibodies that have a human structure.

Methods for preparing and isolating polyclonal and monoclonal antibodiesare well known in the art. See, for example, Cooligan, et al. (eds.),Current Protocols in Immunology, National Institutes of Health, JohnWiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: ALaboratory Manual, second edition, Cold Spring Harbor, N.Y., 1989; andHurrell, J. G. R. (ed.), Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Inc., Boca Raton, Fla., 1982. As would beevident to one of ordinary skill in the art, polyclonal antibodies canbe generated from inoculating a variety of warm-blooded animals such ashorses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats witha zvegf4 polypeptide or a fragment thereof. The immunogenicity of azvegf4 polypeptide may be increased through the use of an adjuvant, suchas alum (aluminum hydroxide) or Freund's complete or incompleteadjuvant. Polypeptides useful for immunization also include fusionpolypeptides, such as fusions of zvegf4 or a portion thereof with animmunoglobulin polypeptide or with maltose binding protein. If thepolypeptide portion is “hapten-like”, such portion may be advantageouslyjoined or linked to a macromolecular carrier (such as keyhole limpethemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) forimmunization.

Alternative techniques for generating or selecting antibodies usefulherein include in vitro exposure of lymphocytes to zvegf4 protein orpeptide, and selection of antibody display libraries in phage or similarvectors (for instance, through use of immobilized or labeled zvegf4protein or peptide). Genes encoding polypeptides having potential zvegf4polypeptide binding domains can be obtained by screening random peptidelibraries displayed on phage (phage display) or on bacteria, such as E.coli. Nucleotide sequences encoding the polypeptides can be obtained ina number of ways, such as through random mutagenesis and randompolynucleotide synthesis. These random peptide display libraries can beused to screen for peptides that interact with a known target, which canbe a protein or polypeptide, such as a ligand or receptor, a biologicalor synthetic macromolecule, or organic or inorganic substance.Techniques for creating and screening such random peptide displaylibraries are known in the art (Ladner et al., U.S. Pat. No. 5,223,409;Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S. Pat. No.5,403,484; and Ladner et al., U.S. Pat. No. 5,571,698), and randompeptide display libraries and kits for screening such libraries areavailable commercially, for instance from Clontech Laboratories (PaloAlto, Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs,Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using the zvegf4sequences disclosed herein to identify proteins which bind to zvegf4.These “binding proteins”, which interact with zvegf4 polypeptides, canbe used for tagging cells or for isolating homologous polypeptides byaffinity purification, or they can be directly or indirectly conjugatedto drugs, toxins, radionuclides, and the like. Binding proteins can alsobe used in analytical methods, such as for screening expressionlibraries and for neutralizing zvegf4 activity; for diagnostic assaysfor determining circulating levels of polypeptides; for detecting orquantitating soluble polypeptides as marker of underlying pathology ordisease; and as zvegf4 antagonists to block zvegf4 binding and signaltransduction in vitro and in vivo.

Antibodies are determined to be specifically binding if they bind to azvegf4 polypeptide, peptide or epitope with an affinity at least 10-foldgreater than the binding affinity to control (non-zvegf4) polypeptide orprotein. In this regard, a “non-zvegf4 polypeptide” includes the relatedmolecules VEGF, VEGF-B, VEGF-C, VEGF-D, zvegf3, PlGF, PDGF-A, andPDGF-B, but excludes zvegf4 polypeptides from non-human species. Due tothe high level of amino acid sequence identity expected between zvegf4orthologs, antibodies specific for human zvegf4 may also bind to zvegf4from other species. The binding affinity of an antibody can be readilydetermined by one of ordinary skill in the art, for example, byScatchard analysis (Scatchard, G., Ann. NY Acad. Sci. 51: 660-672,1949). Methods for screening and isolating specific antibodies are wellknown in the art. See, for example, Paul (ed.), Fundamental Immunology,Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43:1-98, 1988;Goding, J. W. (ed.), Monoclonal Antibodies: Principles and Practice,Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2:67-101,1984.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to zvegf4 proteins orpeptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutant zvegf4protein or polypeptide.

Of particular interest are neutralizing antibodies, that is antibodiesthat block zvegf4 biological activity. Within the present invention, anantibody is considered to be neutralizing if the antibody blocks atleast 50% of the biological activity of a zvegf4 protein when theantibody is present in a 1000-fold molar excess. Within certainembodiments of the invention the antibody will neutralize 50% ofbiological activity when present in a 100-fold molar excess or in a10-fold molar excess. Within other embodiments the antibody neutralizesat least 60% of zvegf4 activity, at least 70% of zvegf4 activity, atleast 80% of zvegf4 activity, or at least 90% of zvegf4 activity.

Antibodies to zvegf4 may be used for tagging cells that express zvegf4;for isolating zvegf4 by affinity purification; for diagnostic assays fordetermining circulating levels of zvegf4 polypeptides; for detecting orquantitating soluble zvegf4 as a marker of underlying pathology ordisease; in analytical methods employing FACS; for screening expressionlibraries; for generating anti-idiotypic antibodies; and as neutralizingantibodies or as antagonists to block zvegf4 activity in vitro and invivo. Suitable direct tags or labels include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like; indirect tags or labels mayfeature use of biotin-avidin or other complement/anti-complement pairsas intermediates. Antibodies may also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications.Moreover, antibodies to zvegf4 or fragments thereof may be used in vitroto detect denatured zvegf4 or fragments thereof in assays, for example,Western Blots or other assays known in the art. Antibodies can also beused to target an attached therapeutic or diagnostic moiety to cellsexpressing zvegf4 or receptors for zvegf4. Experimental data suggestthat zvegf4 may bind PDGF alpha and/or beta receptors.

Anti-zvegf4 antibodies may be administered to recipients that wouldbenefit from a decrease in bone proliferation or differentiation, suchas those recipients suffering from osteosarcoma or osteopetrosis. Inanimals overexpressing zvegf4, histological analysis showedproliferation of endosteal bone (particularly in trabecular bone) thatin some instances replaced most of the bone marrow, as well aproliferation of stromal cells in bone. Anti-zvegf4 antibodies wouldinterfere with these processes, and/or would diminish osteoblastproliferation and bone growth stimulation. Anti-zvegf4 antibodies mayalso be used to antagonize production of cartilage by interfering withthe ability of zvegf4 to stimulate the development or proliferation ofchondrocytes.

In addition, anti-zvegf4 antibodies may be used to diminish pro-fibroticresponses. Histological analysis of animals overexpressing zvegf4detected pro-fibrotic responses in certain organs, particularly liver,kidney and lung. Several diseases or conditions involve fibrosis inliver, lung and kidney. More particularly, alcoholism and viralhepatitis generally involve liver fibrosis, which is often a precursorto cirrhosis, which in turn may lead to an irreversible state of liverfailure. Lung fibrosis resulting from exposure to environmental agents(e.g., asbestosis, silicosis) will often manifest as alveolitis orinterstitial inflammation. Also, lung fibrosis may occur as a sideeffect of some cancer therapies, such as ionizing radiation orchemotherpeutic agents. Further, collagen vascular diseases, such asscleroderma and lupus, may also lead to lung fibrosis. In the kidney,the human condition of membranoproliferative glomerulonephritis maycorrespond to the pro-fibrotic response observed in animalsoverexpressing zvegf4. Chronic immune complex deposition, as seen inlupus, hepatitis B and C, and chronic abscesses, may also lead topro-fibrotic responses in the kidney. Administration of anti-zvegf4antibodies may beneficially interfere with zvegf4-stimulatedpro-fibrotic responses. Such responses include: sclerosing peritonitis,adhesions following surgery, particularly laparoscopic surgery, andrestenosis.

Activity of zvegf4 proteins can be measured in vitro using culturedcells or in vivo by administering molecules of the claimed invention toan appropriate animal model. Target cells for use in zvegf4 activityassays include vascular cells (especially endothelial cells, pericytesand smooth muscle cells), hematopoietic (myeloid and lymphoid) cells,liver cells (including hepatocytes, fenestrated endothelial cells,Kupffer cells, and Ito cells), fibroblasts (including human dermalfibroblasts and lung fibroblasts), neurite cells (including astrocytes,glial cells, dendritic cells, and PC-12 cells), fetal lung cells,articular synoviocytes, pericytes, chondrocytes, osteoblasts, kidneymesangial cells, bone marrow stromal cells (see K. Satomura et al., J.Cell. Physiol. 177:426-38, 1998), and other cells having cell-surfacePDGF receptors.

Zvegf4 proteins can be analyzed for receptor binding activity by avariety of methods well known in the art, including receptor competitionassays (Bowen-Pope and Ross, Methods Enzymol. 109:69-100, 1985), use ofsoluble receptors, and use of receptors produced as IgG fusion proteins(U.S. Pat. No. 5,750,375). Receptor binding assays can be performed oncell lines that contain known cell-surface receptors for evaluation. Thereceptors can be naturally present in the cell, or can be recombinantreceptors expressed by genetically engineered cells. Cell types that areable to bind zvegf4 can be identified through the use of a zvegf4polypeptide conjugated to a cytotoxin or other detectable molecule.Suitable detectable molecules include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles, and the like. Suitable cytotoxic moleculesinclude bacterial or plant toxins (for instance, diphtheria toxin,Pseudomonas exotoxin, ricin, abrin, saporin, and the like), as well astherapeutic radionuclides, such as iodine-131, rhenium-188 oryttrium-90. These can be either directly attached to the polypeptide orindirectly attached according to known methods, such as through achelating moiety. Polypeptides can also be conjugated to cytotoxicdrugs, such as adriamycin. For indirect attachment of a detectable orcytotoxic molecule, the detectable or cytotoxic molecule may beconjugated with a member of a complementary/anticomplementary pair,where the other member is bound to the polypeptide or antibody portion.For these purposes, biotin/streptavidin is an exemplarycomplementary/anticomplementary pair. Binding of a zvegf4-toxinconjugate by cells, either in tissue culture, in organ culture, or invivo will allow for the incorporation of the conjugate into the cell,causing cell death. This activity can be used to identify cell typesthat are able to bind and internalize zvegf4. In addition to allowingfor the identification of responsive cell types, toxin conjugates can beused in in vivo studies to identify organs and tissues where zvegf4 hasa biological activity by looking for pathology within the animalfollowing injection of the conjugate.

Activity of zvegf4 proteins can be measured in vitro using culturedcells. Mitogenic activity can be measured using known assays, including³H-thymidine incorporation assays (as disclosed by, e.g., Raines andRoss, Methods Enzymol. 109:749-773, 1985 and Wahl et al., Mol. Cell.Biol. 8:5016-5025, 1988), dye incorporation assays (as disclosed by, forexample, Mosman, J. Immunol. Meth. 65:55-63, 1983 and Raz et al., ActaTrop. 68:139-147, 1997) or cell counts. Exemplary mitogenesis assaysmeasure incorporation of ³H-thymidine into (1) 20% confluent cultures tolook for the ability of zvegf4 proteins to further stimulateproliferating cells, and (2) quiescent cells held at confluence for 48hours to look for the ability of zvegf4 proteins to overcomecontact-induced growth inhibition. See also, Gospodarowicz et al., J.Cell. Biol. 70:395-405, 1976; Ewton and Florini, Endocrinol.106:577-583, 1980; and Gospodarowicz et al., Proc. Natl. Acad. Sci. USA86:7311-7315, 1989. Cell differentiation can be assayed using suitableprecursor cells that can be induced to differentiate into a more maturephenotype. For example, endothelial cells and hematopoietic cells arederived from a common ancestral cell, the hemangioblast (Choi et al.,Development 125:725-732, 1998). Mesenchymal stem cells can also be usedto measure the ability of zvegf4 protein to stimulate differentiationinto osteoblasts. Differentiation is indicated by the expression ofosteocalcin, the ability of the cells to mineralize, and the expressionof alkaline phosphatase, all of which can be measured by routine methodsknown in the art. Effects of zvegf4 proteins on tumor cell growth andmetastasis can be analyzed using the Lewis lung carcinoma model, forexample as described by Cao et al., J. Exp. Med. 182:2069-2077, 1995.Activity of zvegf4 proteins on cells of neural origin can be analyzedusing assays that measure effects on neurite growth. Zvegf4 can also beassayed in an aortic ring outgrowth assay (Nicosia and Ottinetti,Laboratory Investigation 63:115, 1990; Villaschi and Nicosia, Am. J.Pathology 143:181-190, 1993).

Zvegf4 activity may also be detected using assays designed to measurezvegf4-induced production of one or more additional growth factors orother macromolecules. Such assays include those for determining thepresence of hepatocyte growth factor (HGF), epidermal growth factor(EGF), transforming growth factor alpha (TGFα), interleukin-6 (IL-6),VEGF, acidic fibroblast growth factor (aFGF), and angiogenin. Suitableassays include mitogenesis assays using target cells responsive to themacromolecule of interest, receptor-binding assays, competition bindingassays, immunological assays (e.g., ELISA), and other formats known inthe art. Metalloprotease secretion is measured from treated primaryhuman dermal fibroblasts, synoviocytes and chondrocytes. The relativelevels of collagenase, gelatinase and stromalysin produced in responseto culturing in the presence of a zvegf4 protein is measured usingzymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96-106,1990). Procollagen/collagen synthesis by dermal fibroblasts andchondrocytes in response to a test protein is measured using ³H-prolineincorporation into nascent secreted collagen. ³H-labeled collagen isvisualized by SDS-PAGE followed by autoradiography (Unemori and Amento,J. Biol. Chem. 265: 10681-10685, 1990). Glycosaminoglycan (GAG)secretion from dermal fibroblasts and chondrocytes is measured using a1,9-dimethylmethylene blue dye binding assay (Farndale et al., Biochim.Biophys. Acta 883:173-177, 1986). Collagen and GAG assays are alsocarried out in the presence of IL-1 or TGF-β to examine the ability ofzvegf4 protein to modify the established responses to these cytokines.

Monocyte activation assays are carried out (1) to look for the abilityof zvegf4 proteins to further stimulate monocyte activation, and (2) toexamine the ability of zvegf4 proteins to modulate attachment-induced orendotoxin-induced monocyte activation (Fuhlbrigge et al., J. Immunol.138: 3799-3802, 1987). IL-1β and TNFα levels produced in response toactivation are measured by ELISA (Biosource, Inc. Camarillo, Calif.).Monocyte/macrophage cells, by virtue of CD14 (LPS receptor), areexquisitely sensitive to endotoxin, and proteins with moderate levels ofendotoxin-like activity will activate these cells.

Hematopoietic activity of zvegf4 proteins can be assayed on varioushematopoietic cells in culture. Suitable assays include primary bonemarrow or peripheral blood leukocyte colony assays, and later stagelineage-restricted colony assays, which are known in the art (e.g.,Holly et al., WIPO Publication WO 95/21920). Marrow cells plated on asuitable semi-solid medium (e.g., 50% methylcellulose containing 15%fetal bovine serum, 10% bovine serum albumin, and 0.6% PSN antibioticmix) are incubated in the presence of test polypeptide, then examinedmicroscopically for colony formation. Known hematopoietic factors areused as controls. Mitogenic activity of zvegf4 polypeptides onhematopoietic cell lines can be measured using ³H-thymidineincorporation assays, dye incorporation assays, or cell counts (Rainesand Ross, Methods Enzymol. 109:749-773, 1985 and Foster et al., U.S.Pat. No. 5,641,655). For example, cells are cultured in multi-wellmicrotiter plates. Test samples and ³H-thymidine are added, and thecells are incubated overnight at 37° C. Contents of the wells aretransferred to filters, dried, and counted to determine incorporation oflabel. Cell proliferation can also be measured using a colorimetricassay based on the metabolic breakdown of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Mosman, ibid.). Briefly, a solution of MTT is added to 100 μl of assaycells, and the cells are incubated at 37° C. After 4 hours, 200 μl of0.04 N HCl in isopropanol is added, the solution is mixed, and theabsorbance of the sample is measured at 570 nm.

Cell migration is assayed essentially as disclosed by Kähler et al.(Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939, 1997). Aprotein is considered to be chemotactic if it induces migration of cellsfrom an area of low protein concentration to an area of high proteinconcentration. The assay is performed using modified Boyden chamberswith a polystryrene membrane separating the two chambers (Transwell;Corning Costar Corp.). The test sample, diluted in medium containing 1%BSA, is added to the lower chamber of a 24-well plate containingTranswells. Cells are then placed on the Transwell insert that has beenpretreated with 0.2% gelatin. Cell migration is measured after 4 hoursof incubation at 37° C. Non-migrating cells are wiped off the top of theTranswell membrane, and cells attached to the lower face of the membraneare fixed and stained with 0.1% crystal violet. Stained cells are thencounted directly using a microscope, or extracted with 10% acetic acidand absorbance is measured at 600 nm. Migration is then calculated froma standard calibration curve.

Cell adhesion activity is assayed essentially as disclosed by LaFleur etal. (J. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter platesare coated with the test protein, non-specific sites are blocked withBSA, and cells (such as smooth muscle cells, leukocytes, or endothelialcells) are plated at a density of approximately 10⁴-10⁵ cells/well. Thewells are incubated at 37° C. (typically for about 60 minutes), thennon-adherent cells are removed by gentle washing. Adhered cells arequantitated by conventional methods (e.g., by staining with crystalviolet, lysing the cells, and determining the optical density of thelysate). Control wells are coated with a known adhesive protein, such asfibronectin or vitronectin.

Assays for angiogenic activity are also known in the art. For example,the effect of zvegf4 proteins on primordial endothelial cells inangiogenesis can be assayed in the chick chorioallantoic membraneangiogenesis assay (Leung, Science 246:1306-1309, 1989; Ferrara, Ann. NYAcad. Sci. 752:246-256, 1995). Briefly, a small window is cut into theshell of an eight-day old fertilized egg, and a test substance isapplied to the chorioallantoic membrane. After 72 hours, the membrane isexamined for neovascularization. Other suitable assays includemicroinjection of early stage quail (Coturnix cotumix japonica) embryosas disclosed by Drake et al. (Proc. Natl. Acad. Sci. USA 92:7657-7661,1995); the rodent model of corneal neovascularization disclosed byMuthukkaruppan and Auerbach (Science 205:1416-1418, 1979), wherein atest substance is inserted into a pocket in the cornea of an inbredmouse; and the hampster cheek pouch assay (Höckel et al., Arch. Surg.128:423-429, 1993). Induction of vascular permeability, which isindicative of angiogenic activity, is measured in assays designed todetect leakage of protein from the vasculature of a test animal (e.g.,mouse or guinea pig) after administration of a test compound (Miles andMiles, J. Physiol. 118:228-257, 1952; Feng et al., J. Exp. Med.183:1981-1986, 1996). In vitro assays for angiogenic activity includethe tridimensional collagen gel matrix model (Pepper et al. Biochem.Biophys. Res. Comm. 189:824-831, 1992 and Ferrara et al., Ann. NY Acad.Sci. 732:246-256, 1995), which measures the formation of tube-likestructures by microvascular endothelial cells; and basement membranematrix models (Grant et al., “Angiogenesis as a component ofepithelial-mesenchymal interactions” in Goldberg and Rosen,Epithelial-Mesenchymal Interaction in Cancer, Birkhäuser Verlag, 1995,235-248; Baatout, Anticancer Research 17:451-456, 1997), which are usedto determine effects on cell migration and tube formation by endothelialcells seeded in a basement membrane extract enriched in laminin (e.g.,Matrigel®; Becton Dickinson, Franklin Lakes, N.J.). Angiogenesis assayscan be carried out in the presence and absence of VEGF to assesspossible combinatorial effects. VEGF can be used as a control within invivo assays.

The activity of zvegf4 proteins, agonists, antagonists, and antibodiesof the present invention can be measured, and compounds screened toidentify agonists and antagonists, using assays that measure axonguidance and growth. Of particular interest are assays that indicatechanges in neuron growth patterns, for example those disclosed inHastings, WIPO Publication WO 97/29189 and Walter et al., Development101:685-96, 1987. Assays to measure the effects on neuron growth arewell known in the art. For example, the C assay (e.g., Raper andKapfhammer, Neuron 4:21-9, 1990 and Luo et al., Cell 75:217-27, 1993)can be used to determine collapsing activity of zvegf4 on growingneurons. Other methods that can assess zvegf4-induced inhibition ofneurite extension or divert such extension are also known. See, Goodman,Anna. Rev. Neurosci. 19:341-77, 1996. Conditioned media from cellsexpressing a zvegf4 protein, a zvegf4 agonist, or a zvegf4 antagonist,or aggregates of such cells, can by placed in a gel matrix near suitableneural cells, such as dorsal root ganglia (DRG) or sympathetic gangliaexplants, which have been co-cultured with nerve growth factor. Comparedto control cells, zvegf4-induced changes in neuron growth can bemeasured (as disclosed by, for example, Messersmith et al., Neuron14:949-59, 1995 and Puschel et al., Neuron 14:941-8, 1995). Likewiseneurite outgrowth can be measured using neuronal cell suspensions grownin the presence of molecules of the present invention. See, for example,O'Shea et al., Neuron 7:231-7, 1991 and DeFreitas et al., Neuron15:333-43, 1995. PC12 Pheochromocytoma cells (see Banker and Goslin, inCulturing Nerve Cells, chapter 6, “Culture and experimental use of thePC12 rat Pheochromocytoma cell line”; also, see Rydel and Greene, J.Neuroscience 7(11): 3639-53, November 1987) can be grown in the presenceof zvegf4 to examine effects on neurite outgrowth. PC12 cellspre-treated with NGF to induce differentiation into a neuronalpopulation can also be exposed to zvegf4 to determine the ability ofzvegf4 to promote survival of neuronal cells.

The biological activities of zvegf4 proteins can be studied in non-humananimals by administration of exogenous protein, by expression ofzvegf4-encoding polynucleotides, and by suppression of endogenous zvegf4expression through antisense or knock-out techniques. Zvegf4 proteinscan be administered or expressed individually, in combination with otherzvegf4 proteins, or in combination with non-vegf3 proteins, includingother growth factors (e.g., other VEGFs, PlGFs, or PDGFs). For example,a combination of zvegf4 polypeptides (e.g., a combination ofzvegf4₁₉₋₁₇₉ and zvegf-4₂₅₈₋₃₇₀) can be administered to a test animal orexpressed in the animal. Test animals are monitored for changes in suchparameters as clinical signs, body weight, blood cell counts, clinicalchemistry, histopathology, and the like.

Stimulation of coronary collateral growth can be measured in knownanimal models, including a rabbit model of peripheral limb ischemia andhind limb ischemia and a pig model of chronic myocardial ischemia(Ferrara et al., Endocrine Reviews 18:4-25, 1997). Zvegf4 proteins areassayed in the presence and absence of VEGFs, angiopoietins, and basicFGF to test for combinatorial effects. These models can be modified bythe use of adenovirus or naked DNA for gene delivery as disclosed inmore detail below, resulting in local expression of the test protein(s).

Efficacy of zvegf4 polypeptides in promoting wound healing can beassayed in animal models. One such model is the linear skin incisionmodel of Mustoe et al. (Science 237:1333, 1987). In a typical procedure,a 6-cm incision is made in the dorsal pelt of an adult rat, then closedwith wound clips. Test substances and controls (in solution, gel, orpowder form) are applied before primary closure. Although administrationis commonly limited to a single application, additional applications canbe made on succeeding days by careful injection at several sites underthe incision. Wound breaking strength is evaluated between 3 and 21 dayspost-wounding. In a second model, multiple, small, full-thicknessexcisions are made on the ear of a rabbit. The cartilage in the earsplints the wound, removing the variable of wound contraction from theevaluation of closure. Experimental treatments and controls are applied.The geometry and anatomy of the wound site allow for reliablequantification of cell ingrowth and epithelial migration, as well asquantitative analysis of the biochemistry of the wounds (e.g., collagencontent). See, Mustoe et al., J. Clin. Invest. 87:694, 1991. The rabbitear model can be modified to create an ischemic wound environment, whichmore closely resembles the clinical situation (Ahn et al., Ann. Plast.Surg. 24:17, 1990). Within a third model, healing of partial-thicknessskin wounds in pigs or guinea pigs is evaluated (LeGrand et al., GrowthFactors 8:307, 1993). Experimental treatments are applied daily on orunder dressings. Seven days after wounding, granulation tissue thicknessis determined. This model is commonly used for dose-response studies, asit is more quantitative than other in vivo models of wound healing. Afull thickness excision model can also be employed. Within this model,the epidermis and dermis are removed down to the panniculus carnosum inrodents or the subcutaneous fat in pigs. Experimental treatments areapplied topically on or under a dressing, and can be applied daily ifdesired. The wound closes by a combination of contraction and cellingrowth and proliferation. Measurable endpoints include time to woundclosure, histologic score, and biochemical parameters of wound tissue.Impaired wound healing models are also known in the art (e.g., Cromacket al., Surgery 113:36, 1993; Pierce et al., Proc. Natl. Acad. Sci. USA86:2229, 1989; Greenhalgh et al., Amer. J. Pathol. 136:1235, 1990).Delay or prolongation of the wound healing process can be inducedpharmacologically by treatment with steroids, irradiation of the woundsite, or by concomitant disease states (e.g., diabetes). Linearincisions or full-thickness excisions are most commonly used as theexperimental wound. Endpoints are as disclosed above for each type ofwound. Subcutaneous implants can be used to assess compounds acting inthe early stages of wound healing (Broadley et al., Lab. Invest. 61:571,1985; Sprugel et al., Amer. J. Pathol. 129: 601, 1987). Implants areprepared in a porous, relatively non-inflammatory container (e.g.,polyethylene sponges or expanded polytetrafluoroethylene implants filledwith bovine collagen) and placed subcutaneously in mice or rats. Theinterior of the implant is empty of cells, producing a “wound space”that is well-defined and separable from the preexisting tissue. Thisarrangement allows the assessment of cell influx and cell type as wellas the measurement of vasculogenesis/angiogenesis and extracellularmatrix production.

Expression of zvegf4 proteins in animals provides models for study ofthe biological effects of overproduction or inhibition of proteinactivity in vivo. Zvegf4-encoding polynucleotides can be introduced intotest animals, such as mice, using viral vectors or naked DNA, ortransgenic animals can be produced. A zvegf4 protein will commonly beexpressed with a secretory peptide. Suitable secretory peptides includethe zvegf4 secretory peptide (e.g., residues 1-18 of SEQ ID NO:2) andheterologous secretory peptides. An exemplary heterologous secretorypeptide is that of human tissue plasminogen activator (t-PA). The t-PAsecretory peptide may be modified to reduce undesired proteolyticcleavage as disclosed in U.S. Pat. No. 5,641,655.

One in vivo approach for assaying proteins of the present inventionutilizes viral delivery systems. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, retroviruses, vaccinia virus, andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acids. For review, see Becker et al., Meth. CellBiol. 43:161-89, 1994; and Douglas and Curiel, Science & Medicine4:44-53, 1997. The adenovirus system offers several advantages.Adenovirus can (i) accommodate relatively large DNA inserts; (ii) begrown to high-titer; (iii) infect a broad range of mammalian cell types;and (iv) be used with many different promoters including ubiquitous,tissue specific, and regulatable promoters. Because adenoviruses arestable in the bloodstream, they can be administered by intravenousinjection.

Using adenovirus vectors where portions of the adenovirus genome aredeleted, inserts are incorporated into the viral DNA by direct ligationor by homologous recombination with a co-transfected plasmid. In anexemplary system, the essential E1 gene has been deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell (the human 293 cell line is exemplary). Whenintravenously administered to intact animals, adenovirus primarilytargets the liver. If the adenoviral delivery system has an E1 genedeletion, the virus cannot replicate in the host cells. However, thehost's tissue (e.g., liver) will express and process (and, if asecretory signal sequence is present, secrete) the heterologous protein.Secreted proteins will enter the circulation in the highly vascularizedliver, and effects on the infected animal can be determined. Intranasaldelivery of adenovirus expressing zvegf4 will target the zvegf4 proteinto lung tissue. Further, adenovirus expressing zvegf4 can beadministered directly into brain tissue. Adenoviral vectors containingvarious deletions of viral genes can be used in an attempt to reduce oreliminate immune responses to the vector. Such adenoviruses are E1deleted, and in addition contain deletions of E2A or E4 (Lusky et al.,J. Virol. 72:2022-2032, 1998; Raper et al., Human Gene Therapy9:671-679, 1998). In addition, deletion of E2b is reported to reduceimmune responses (Amalfitano, et al., J. Virol. 72:926-933, 1998).Generation of so-called “gutless” adenoviruses where all viraltranscription units are deleted is particularly advantageous forinsertion of large inserts of heterologous DNA. For review, see Yeh andPerricaudet, FASEB J. 11:615-623, 1997.

In another embodiment, a zvegf4 gene can be introduced in a retroviralvector as described, for example, by Anderson et al., U.S. Pat. No.5,399,346; Mann et al., Cell 33:153, 1983; Temin et al., U.S. Pat. No.4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J.Virol. 62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263; Doughertyet al., WIPO publication WO 95/07358; and Kuo et al., Blood 82:845,1993.

In an alternative method, the vector can be introduced by “lipofection”in vivo using liposomes. Synthetic cationic lipids can be used toprepare liposomes for in vivo transfection of a gene encoding a marker(Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey etal., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988). The use oflipofection to introduce exogenous genes into specific organs in vivohas certain practical advantages. Molecular targeting of liposomes tospecific cells represents one area of benefit. For instance, directingtransfection to particular cell types is particularly advantageous in atissue with cellular heterogeneity, such as the pancreas, liver, kidney,and brain. Lipids may be chemically coupled to other molecules for thepurpose of targeting. Targeted peptides (e.g., hormones orneurotransmitters), proteins such as antibodies, or non-peptidemolecules can be coupled to liposomes chemically.

Within another embodiment target cells are removed from the animal, andthe DNA is introduced as a naked DNA plasmid. The transformed cells arethen re-implanted into the body of the animal. Naked DNA vectors can beintroduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

Mice engineered to express the zvegf4 gene, referred to as “transgenicmice,” and mice that exhibit a complete absence of zvegf4 gene function,referred to as “knockout mice,” can also be generated (Snouwaert et al.,Science 257:1083, 1992; Lowell et al., Nature 366:740-42, 1993;Capecchi, Science 244:1288-1292, 1989; Palmiter et al., Ann. Rev. Genet.20:465-499, 1986). Transgenesis experiments can be performed usingnormal mice or mice with genetic disease or other altered phenotypes.Transgenic mice that over-express zvegf4, either ubiquitously or under atissue-specific or tissue-restricted promoter, can be used to determinewhether or not over-expression causes a phenotypic change. Exemplarypromoters include metallothionein, albumin, ApoA1 and enolase genepromoters. The metallothionein-1 (MT-1) promoter provides expression inliver and other tissues, often leading to high levels of circulatingprotein. Over-expression of a wild-type zvegf4 polypeptide, polypeptidefragment or a mutant thereof may alter normal cellular processes,resulting in a phenotype that identifies a tissue in which zvegf4expression is functionally relevant and may indicate a therapeutictarget for the zvegf4, its agonists or antagonists. For example, atransgenic mouse can be engineered to over-expresses a full-lengthzvegf4 sequence, which may result in a phenotype that shows similaritywith human diseases. Similarly, knockout zvegf4 mice can be used todetermine where zvegf4 is absolutely required in vivo. The phenotype ofknockout mice is predictive of the in vivo effects of zvegf4antagonists. Knockout mice can also be used to study the effects ofzvegf4 proteins in models of disease, including, for example, cancer,atherosclerosis, rheumatoid arthritis, ischemia, and cardiovasculardisease. The human zvegf4 cDNA can be used to isolate murine zvegf4mRNA, cDNA and genomic DNA as disclosed above, which are subsequentlyused to generate knockout mice. These mice may be employed to study thezvegf4 gene and the protein encoded thereby in an in vivo system, andcan be used as in vivo models for corresponding human diseases.Moreover, transgenic mice expressing zvegf4 antisense polynucleotides orribozymes directed against zvegf4, described herein, can be usedanalogously to knockout mice described above.

Antisense methodology can be used to inhibit zvegf4 gene transcriptionto examine the effects of such inhibition in vivo. Polynucleotides thatare complementary to a segment of a zvegf4-encoding polynucleotide(e.g., a polynucleotide as set froth in SEQ ID NO:1) are designed tobind to zvegf4-encoding mRNA and to inhibit translation of such mRNA.Such antisense oligonucleotides can also be used to inhibit expressionof zvegf4 polypeptide-encoding genes in cell culture.

Zvegf4 proteins may be used therapeutically in human and veterinarymedicine to stimulate tissue development or repair, or cellulardifferentiation or proliferation. Specific applications include, withoutlimitation: the treatment of full-thickness skin wounds, includingvenous stasis ulcers and other chronic, non-healing wounds, particularlyin cases of compromised wound healing due to diabetes mellitus,connective tissue disease, smoking, burns, and other exacerbatingconditions; fracture repair; skin grafting; within reconstructivesurgery to promote neovascularization and increase skin flap survival;to establish vascular networks in transplanted cells and tissues, suchas transplanted islets of Langerhans; to treat female reproductive tractdisorders, including acute or chronic placental insufficiency (animportant factor causing perinatal morbidity and mortality) andprolonged bleeding; to promote the growth of tissue damaged byperiodontal disease; to promote endothelialization of vascular graftsand stents; in the treatment of acute and chronic lesions of thegastrointestinal tract, including duodenal ulcers, which arecharacterized by a deficiency of microvessels; to promote angiogenesisand prevent neuronal degeneration due to acute or chronic cerebralischemia; to accelerate the formation of collateral blood vessels inischemic limbs; to promote vessel re-endothelialization and to reduceintimal hyperplasia following invasive procedures such as balloonangioplasty and stent placement; to promote vessel repair anddevelopment of collateral circulation following myocardial infarction soas to limit ischemic injury; and to stimulate hematopoiesis. Thepolypeptides are also useful additives in tissue adhesives for promotingrevascularization of the healing tissue.

Of particular interest is the use of zvegf4 for the treatment or repairof liver damage, including damage due to chronic liver disease,including chronic active hepatitis and many other types of cirrhosis.Widespread, massive necrosis, including destruction of virtually theentire liver, can be caused by, inter alia, fulminant viral hepatitis;overdoses of the analgesic acetaminophen; exposure to other drugs andchemicals such as halothane, monoamine oxidase inhibitors, agentsemployed in the treatment of tuberculosis, phosphorus, carbontetrachloride, and other industrial chemicals. Conditions associatedwith ultrastructural lesions that do not necessarily produce obviousliver cell necrosis include Reye's syndrome in children, tetracyclinetoxicity, and acute fatty liver of pregnancy. Cirrhosis, a diffuseprocess characterized by fibrosis and a conversion of normalarchitecture into structurally abnormal nodules, can come about for avariety reasons including alcohol abuse, post necrotic cirrhosis(usually due to chronic active hepatitis), biliary cirrhosis, pigmentcirrhosis, cryptogenic cirrhosis, Wilson's disease, andalpha-1-antitrypsin deficiency. Zvegf4 may also be useful for thetreatment of hepatic chronic passive congestion (CPC) and centralhemorrhagic necrosis (CHN), which are two circulatory changesrepresenting a continuum encountered in right-sided heart failure. Othercirculatory disorders that may be treated with zvegf4 include hepaticvein thrombosis, portal vein thrombosis, and cardiac sclerosis. In casesof liver fibrosis, it may be beneficial to administer a zvegf4antagonist to suppress the activation of stellate cells, which have beenimplicated in the production of extracellular matrix in fibrotic liver(Li and Friedman, J. Gastroenterol. Hepatol. 14:618-633, 1999). Moregenerally, zvegf4 may be beneficially used as an anti-fibrotic agent.Conditions that are characterized by a pro-fibrotic response includesclerosing peritonitis; adhesions following surgery (particularlylaparoscopic surgery), which may lead to small bowel obstruction,difficulties on re-operation, pelvic adhesions and pelvic pain (see N.Panay and A. M. Lower, Curr. Opin. Obstet. Gynecol. 11:379-85, 1999);pulmonary fibrosis; kidney fibrosis; and restenosis.

Zvegf4 polypeptides can be administered alone or in combination withother vasculogenic or angiogenic agents, including VEGF andangiopoietins 1 and 2. For example, basic and acidic FGFs, Ang-1, Ang-2,and VEGF have been found to play a role in the development of collateralcirculation, and the combined use of zvegf4 with one or more of thesefactors may be advantageous. VEGF has also been implicated in thesurvival of transplanted islet cells (Gorden et al. Transplantation63:436-443, 1997; Pepper, Arteriosclerosis, Throm. and Vascular Biol.17:605-619, 1997). Basic FGF has been shown to induce angiogenesis andaccelerate healing of ulcers in experimental animals (reviewed byFolkman, Nature Medicine 1:27-31, 1995). VEGF has been shown to promotevessel re-endothelialization and to reduce intimal hyperplasia in animalmodels of restenosis (Asahara et al., Circulation 91:2802-2809, 1995;Callow et al., Growth Factors 10:223-228, 1994); efficacy of zvegf4polypeptides can be tested in these and other known models. When usingzvegf4 in combination with an additional agent, the two compounds can beadministered simultaneously or sequentially as appropriate for thespecific condition being treated.

Zvegf4 proteins may be used either alone or in combination with otherhematopoietic factors such as IL-3, G-CSF, GM-CSF, or stem cell factorto enhance expansion and mobilization of hematopoietic stem cells,including endothelial precursor stem cells. Cells that can be expandedin this manner include cells isolated from bone marrow, including bonemarrow stromal cells (see K. Satomura et al., J. Cell. Physiol.177:426-38, 1998), or cells isolated from blood. Zvegf4 proteins mayalso be given directly to an individual to enhance endothelial stem cellproduction and differentiation within the treated individual. The stemcells, either developed within the patient, or provided back to apatient, may then play a role in modulating areas of ischemia within thebody, thereby providing a therapeutic effect. These cells may also beuseful in enhancing re-endothelialization of areas devoid of endothelialcoverage, such as vascular grafts, vascular stents, and areas where theendothelial coverage has been damaged or removed (e.g., areas ofangioplasty). Zvegf4 proteins may also be used in combination with othergrowth and differentiation factors such as angiopoietin-1 (Davis et al.,Cell 87:1161-1169, 1996) to help create and stabilize new vesselformation in areas requiring neovascularization, including areas ofischemia (cardiac or peripheral ischemia), organ transplants, woundhealing, and tissue grafting.

Zvegf4 proteins, agonists and antagonists may be used to modulateneurite growth and development and demarcate nervous system structures.As such, Zvegf4 proteins, agonists, and antagonists would be useful as atreatment of peripheral neuropathies by increasing spinal cord andsensory neurite outgrowth. A zvegf4 antagonist could be part of atherapeutic treatment for the regeneration of neurite outgrowthsfollowing strokes, brain damage caused by head injuries and paralysiscaused by spinal injuries. Application may also be made in treatingneurodegenerative diseases such as multiple sclerosis, Alzheimer'sdisease and Parkinson's disease. Application may also be made inmediating development and innervation pattern of stomach tissue.

Zvegf4 has been found to have PDGF-like activity, including mitogenicactivity on fibroblasts, vascular smooth muscle cells, and pericytes.Zvegf4 has also been found to stimulate bone growth in an animal model.These results suggest that zvegf4 proteins will be useful in promotingthe growth of bone and ligament. Such uses include, for example,treatment of periodontal disease, fractures (including non-unionfractures), implant recipient sites, bone grafts, and joint injuriesinvolving cartilage and/or ligament damage. Zvegf4 may be used incombination with other bone stimulating factors, such as IGF-1, EGF,TGF-β, PDGF, and BMPs. Methods for using growth factors in the treatmentof periodontal disease are known in the art. See, for example, U.S. Pat.No. 5,124,316 and Lynch et al., ibid.

For pharmaceutical use, zvegf4 proteins, antagonist, and antibodies areformulated for topical or parenteral, particularly intravenous orsubcutaneous, delivery according to conventional methods. In general,pharmaceutical formulations will include a zvegf4 polypeptide incombination with a pharmaceutically acceptable vehicle, such as saline,buffered saline, 5% dextrose in water, or the like. Formulations mayfurther include one or more excipients, preservatives, solubilizers,buffering agents, albumin to prevent protein loss on vial surfaces,thickeners, gelling agents, etc. Methods of formulation are well knownin the art and are disclosed, for example, in Remington: The Science andPractice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, Pa.,19th ed., 1995. Zvegf4 will ordinarily be used in a concentration ofabout 10 to 100 μg/ml of total volume, although concentrations in therange of 1 ng/ml to 1000 μg/ml may be used. For topical application,such as for the promotion of wound healing, the protein will be appliedin the range of 0.1-10 μg/cm² of wound area, with the exact dosedetermined by the clinician according to accepted standards, taking intoaccount the nature and severity of the condition to be treated, patienttraits, etc. Determination of dose is within the level of ordinary skillin the art. The therapeutic formulations will generally be administeredover the period required for neovascularization, typically from one toseveral months and, in treatment of chronic conditions, for a year ormore. Dosing is daily or intermittently over the period of treatment.Intravenous administration will be by bolus injection or infusion over atypical period of one to several hours. Sustained release formulationscan also be employed. In general, a therapeutically effective amount ofzvegf4 is an amount sufficient to produce a clinically significantchange in the treated condition, such as a clinically significantreduction in time required by wound closure, a significant reduction inwound area, a significant improvement in vascularization, a significantreduction in morbidity, or a significantly increased histological score.

Proteins of the present invention are useful for modulating theproliferation, differentiation, migration, or metabolism of responsivecell types, which include both primary cells and cultured cell lines. Ofparticular interest in this regard are hematopoietic cells (includingstem cells and mature myeloid and lymphoid cells), endothelial cells,neuronal cells, mesenchymal cells (including fibroblasts, pericytes,stellate cells, mesangial cells, chondrocytes and smooth muscle cells),and bone-derived cells (including osteoblast and osteoclast precursors).Zvegf4 polypeptides are added to tissue culture media for these celltypes at a concentration of about 10 μg/ml to about 1000 ng/ml. Thoseskilled in the art will recognize that zvegf4 proteins can beadvantageously combined with other growth factors in culture media.

Within the laboratory research field, zvegf4 proteins can also be usedas molecular weight standards; as reagents in assays for determiningcirculating levels of the protein, such as in the diagnosis of disorderscharacterized by over- or under-production of zvegf4 protein; or asstandards in the analysis of cell phenotype.

Zvegf4 proteins can also be used to identify inhibitors of theiractivity. Test compounds are added to the assays disclosed above toidentify compounds that inhibit the activity of zvegf4 protein. Inaddition to those assays disclosed above, samples can be tested forinhibition of zvegf4 activity within a variety of assays designed tomeasure receptor binding or the stimulation/inhibition ofzvegf4-dependent cellular responses. For example, zvegf4-responsive celllines can be transfected with a reporter gene construct that isresponsive to a zvegf4-stimulated cellular pathway. Reporter geneconstructs of this type are known in the art, and will generallycomprise a zvegf4-activated serum response element (SRE) operably linkedto a gene encoding an assayable protein, such as luciferase. Candidatecompounds, solutions, mixtures or extracts are tested for the ability toinhibit the activity of zvegf4 on the target cells as evidenced by adecrease in zvegf4 stimulation of reporter gene expression. Assays ofthis type will detect compounds that directly block zvegf4 binding tocell-surface receptors, as well as compounds that block processes in thecellular pathway subsequent to receptor-ligand binding. In thealternative, compounds or other samples can be tested for directblocking of zvegf4 binding to receptor using zvegf4 tagged with adetectable label (e.g., ¹²⁵I, biotin, horseradish peroxidase, FITC, andthe like). Within assays of this type, the ability of a test sample toinhibit the binding of labeled zvegf4 to the receptor is indicative ofinhibitory activity, which can be confirmed through secondary assays.Receptors used within binding assays may be cellular receptors orisolated, immobilized receptors.

The activity of zvegf4 proteins can be measured with a silicon-basedbiosensor microphysiometer that measures the extracellular acidificationrate or proton excretion associated with receptor binding and subsequentphysiologic cellular responses. An exemplary such device is theCytosensor™ Microphysiometer manufactured by Molecular Devices,Sunnyvale, Calif. A variety of cellular responses, such as cellproliferation, ion transport, energy production, inflammatory response,regulatory and receptor activation, and the like, can be measured bythis method. See, for example, McConnell et al., Science 257:1906-1912,1992; Pitchford et al., Meth. Enzymol. 228:84-108, 1997; Arimilli etal., J. Immunol. Meth. 212:49-59, 1998; and Van Liefde et al., Eur. J.Pharmacol. 346:87-95, 1998. The microphysiometer can be used forassaying adherent or non-adherent eukaryotic or prokaryotic cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including zvegf4 proteins, their agonists, and antagonists. Themicrophysiometer can be used to measure responses of a zvegf4-responsiveeukaryotic cell, compared to a control eukaryotic cell that does notrespond to zvegf4 polypeptide. Zvegf4-responsive eukaryotic cellscomprise cells into which a receptor for zvegf4 has been transfectedcreating a cell that is responsive to zvegf4, as well as cells naturallyresponsive to zvegf4 such as cells derived from vascular or neuraltissue. Differences, measured by a change in extracellularacidification, in the response of cells exposed to zvegf4 polypeptiderelative to a control not exposed to zvegf4, are a direct measurement ofzvegf4-modulated cellular responses. Moreover, such zvegf4-modulatedresponses can be assayed under a variety of stimuli. The presentinvention thus provides methods of identifying agonists and antagonistsof zvegf4 proteins, comprising providing cells responsive to a zvegf4polypeptide, culturing a first portion of the cells in the absence of atest compound, culturing a second portion of the cells in the presenceof a test compound, and detecting a change in a cellular response of thesecond portion of the cells as compared to the first portion of thecells. The change in cellular response is shown as a measurable changein extracellular acidification rate. Culturing a third portion of thecells in the presence of a zvegf4 protein and the absence of a testcompound provides a positive control for the zvegf4-responsive cells anda control to compare the agonist activity of a test compound with thatof the zvegf4 polypeptide. Antagonists of zvegf4 can be identified byexposing the cells to zvegf4 protein in the presence and absence of thetest compound, whereby a reduction in zvegf4-stimulated activity isindicative of antagonist activity in the test compound.

Zvegf4 proteins can also be used to identify cells, tissues, or celllines that respond to a zvegf4-stimulated pathway. The microphysiometer,described above, can be used to rapidly identify ligand-responsivecells, such as cells responsive to zvegf4 proteins. Cells are culturedin the presence or absence of zvegf4 polypeptide. Those cells thatelicit a measurable change in extracellular acidification in thepresence of zvegf4 are responsive to zvegf4. Responsive cells can thanbe used to identify antagonists and agonists of zvegf4 polypeptide asdescribed above.

Inhibitors of zvegf4 activity (zvegf4 antagonists) include anti-zvegf4antibodies and soluble zvegf4 receptors, as well as other peptidic andnon-peptidic agents, including ribozymes, small molecule inhibitors, andangiogenically or mitogenically inactive receptor-binding fragments ofzvegf4 polypeptides. Such antagonists can be use to block biologicalactivities of zvegf4, including mitogenic, chemotactic, or angiogeniceffects. These antagonists are therefore useful in reducing the growthof solid tumors by inhibiting neovascularization of the developing tumoror by directly blocking tumor cell growth; in the treatment of diabeticretinopathy, psoriasis, arthritis, and scleroderma; and in reducingfibrosis, including scar formation. Inhibitors of zvegf4 may also beuseful in the treatment of proliferative vascular disorders whereinzvegf4 activity is pathogenic. Such disorders may includeatherosclerosis and intimal hyperplastic restenosis followingangioplasty, endarterectomy, vascular grafting, organ transplant, orvascular stent emplacement. These conditions involve complex growthfactor-mediated responses wherein certain factors may be beneficial tothe clinical outcome and others may be pathogenic.

Inhibitors of zvegf4 may also prove useful in the treatment of ocularneovascularization, including diabetic retinopathy and age-relatedmacular degeneration. Experimental evidence suggests that theseconditions result from the expression of angiogenic factors induced byhypoxia in the retina.

Zvegf4 antagonists are also of interest in the treatment of inflammatorydisorders, such as rheumatoid arthritis and psoriasis. In rheumatoidarthritis, studies suggest that VEGF plays an important role in theformation of pannus, an extensively vascularized tissue that invades anddestroys cartilage. Psoriatic lesions are hypervascular and overexpressthe angiogenic polypeptide IL-8.

Zvegf4 antagonists may also prove useful in the treatment of infantilehemangiomas, which exhibit overexpression of VEGF and bFGF during theproliferative phase.

Inhibitors are formulated for pharmaceutical use as generally disclosedabove, taking into account the precise chemical and physical nature ofthe inhibitor and the condition to be treated. The relevantdeterminations are within the level of ordinary skill in the formulationart. Other angiogenic and vasculogenic factors, including VEGF and bFGF,have been implicated in pathological neovascularization. In suchinstances it may be advantageous to combine a zvegf4 inhibitor with oneor more inhibitors of these other factors.

The polypeptides, nucleic acids, and antibodies of the present inventionmay be used in diagnosis or treatment of disorders associated with cellloss or abnormal cell proliferation (including cancer), includingimpaired or excessive vasculogenesis or angiogenesis, and diseases ofthe nervous system. Labeled zvegf4 polypeptides may be used for imagingtumors or other sites of abnormal cell proliferation. Becauseangiogenesis in adult animals is generally limited to wound healing andthe female reproductive cycle, it is a very specific indicator ofpathological processes. Angiogenesis is indicative of, for example,developing solid tumors, retinopathies, and arthritis.

Zvegf4 polypeptides and anti-zvegf4 antibodies can be directly orindirectly conjugated to drugs, toxins, radionuclides and the like, andthese conjugates used for in vivo diagnostic or therapeuticapplications. For instance, polypeptides or antibodies of the presentinvention may be used to identify or treat tissues or organs thatexpress a corresponding anti-complementary molecule (receptor orantigen, respectively, for instance). More specifically, zvegf4polypeptides or anti-zvegf4 antibodies, or bioactive fragments orportions thereof, can be coupled to detectable or cytotoxic moleculesand delivered to a mammal having cells, tissues, or organs that expressthe anti-complementary molecule. For example, the CUB domain of zvegf4can be used to target peptidic and non-peptidic moieties to semaphorinsas disclosed above. In another embodiment, polypeptide-toxin fusionproteins or antibody/fragment-toxin fusion proteins may be used fortargeted cell or tissue inhibition or ablation, such as in cancertherapy. Of particular interest in this regard are conjugates of azvegf4 polypeptide and a cytotoxin, which can be used to target thecytotoxin to a tumor or other tissue that is undergoing undesiredangiogenesis or neovascularization.

In another embodiment, zvegf4-cytokine fusion proteins orantibody/fragment-cytokine fusion proteins may be used for enhancing invitro cytotoxicity (for instance, that mediated by monoclonal antibodiesagainst tumor targets) and for enhancing in vivo killing of targettissues (for example, blood and bone marrow cancers). See, generally,Hornick et al., Blood 89:4437-4447, 1997). In general, cytokines aretoxic if administered systemically. The described fusion proteins enabletargeting of a cytokine to a desired site of action, such as a cellhaving binding sites for zvegf4, thereby providing an elevated localconcentration of cytokine. Suitable cytokines for this purpose include,for example, interleukin-2 and granulocyte-macrophage colony-stimulatingfactor (GM-CSF). Such fusion proteins may be used to causecytokine-induced killing of tumors and other tissues undergoingangiogenesis or neovascularization.

In yet another embodiment, a zvegf4 polypeptide or anti-zvegf4 antibodycan be conjugated with a radionuclide, particularly with a beta-emittingor gamma-emitting radionuclide, and used to reduce restenosis. Forinstance, iridium-192 impregnated ribbons placed into stented vessels ofpatients until the required radiation dose was delivered resulted indecreased tissue growth in the vessel and greater luminal diameter thanthe control group, which received placebo ribbons. Further,revascularisation and stent thrombosis were significantly lower in thetreatment group. Similar results are predicted with targeting of abioactive conjugate containing a radionuclide, as described herein.

The bioactive polypeptide or antibody conjugates described herein can bedelivered intravenously, intra-arterially or intraductally, or may beintroduced locally at the intended site of action.

Polynucleotides encoding zvegf4 polypeptides are useful within genetherapy applications where it is desired to increase or inhibit zvegf4activity. For example, Isner et al., The Lancet (ibid.) reported thatVEGF gene therapy promoted blood vessel growth in an ischemic limb.Additional applications of zvegf4 gene therapy include stimulation ofwound healing, repopulation of vascular grafts, stimulation of neuritegrowth, and inhibition of cancer growth and metastasis. Gene deliverysystems useful in this regard include adenovirus, adeno-associatedvirus, and naked DNA vectors.

The present invention also provides polynucleotide reagents fordiagnostic use. For example, a zvegf4 gene, a probe comprising zvegf4DNA or RNA, or a subsequence thereof can be used to determine if amutation has occurred at the zvegf4 locus on human chromosome 11.Detectable chromosomal aberrations at the zvegf4 gene locus include, butare not limited to, aneuploidy, gene copy number changes, insertions,deletions, restriction site changes and rearrangements. Such aberrationscan be detected using polynucleotides of the present invention byemploying molecular genetic techniques, such as restriction fragmentlength polymorphism (RFLP) analysis, short tandem repeat (STR) analysisemploying PCR techniques, and other genetic linkage analysis techniquesknown in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; A. J.Marian, Chest 108:255-265, 1995).

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1

Human Multiple Tissue Northern Blots I, II, III and Human RNA MasterBlots (Clontech Laboratories, Inc., Palo Alto, Calif.) were probed todetermine the tissue expression of zvegf4. Blots were prehybridized for3 hours at 65 degrees in 10 ml of a hybridization solution (ExpressHyb™Hybridization Solution; Clontech Laboratories, Inc.) containing 1 mg ofsalmon sperm DNA that had been boiled 5 minutes, then iced 1 minute. Theprobe used was a 251-bp PCR fragment generated with 20 pmole each ofprimers ZC21,119 (SEQ ID NO:25) and ZC21,120 (SEQ ID NO:26), and 5 μl ofa heart cDNA library prepared from heart RNA using a commerciallyavailable kit (Marathon™ cDNA Amplification Kit from ClontechLaboratories, Inc.). The reaction was run as follows: 94 degrees for 1minute; then 30 cycles of 94 degrees, 20 seconds; 67 degrees, 1 minute;and ended with a 5-minute incubation at 72 degrees. The PCR product wasgel-purified, and the DNA was eluted from the gel slab with a spincolumn containing a silica gel membrane (QIAquick™ Gel Extraction Kit;Qiagen, Inc., Valencia, Calif.).

51 ng of the resulting zvegf4 fragment was labeled with ³²P using acommercially available kit (Rediprime™ II random-prime labeling system;Amersham Pharmacia Biotech, Piscataway, N.J.). Unincorporatedradioactivity was removed with a push column (NucTrap® column;Stratagene, La Jolla, Calif.; see U.S. Pat. No. 5,336,412). 10×10⁶ cpmof the resulting labeled probe and 1 mg of salmon sperm DNA were boiled5 minutes, iced 1 minute, then mixed with 10 ml hybridization solution(ExpressHyb™) and added to blots. Hybridization took place overnight at65 degrees, followed by a wash in 2×SSC, 0.1% SDS at room temperature,followed by a wash in 0.1×SSC, 0.1% SDS at 50 degrees. Blots wereexposed to film at −80 degrees overnight.

There was an approximately 4.4 kb transcript in every tissue except bonemarrow. Heart, pancreas, stomach and adrenal gland showed the strongestzvegf4 expression on the Northern blots, and the dot blot additionallyshowed strong expression in the pituitary gland and the ovary.

Example 2

Zvegf4 was identified from the sequence of a clone from a human chronicmyelogenous leukemia cell (K562) library by its homology to the VEGFfamily. Additional sequence was elucidated from a long sequence read ofa clone from a pituitary library. An antisense expressed sequence tag(EST) for zvegf4 was found, for which its 5′ partner was identified.This 5′ EST (EST448186; GenBank) appeared to contain the 5′ untranslatedsequence for zvegf4. A primer was designed from EST448186 to close thegap in the sequence. 20 pm each of ZC21,987 (SEQ ID NO:27) and ZC21,120(SEQ ID NO:26) and 1.93 lag of a thyroid library were used in the PCRreaction. It was a modified PCR reaction using 5% DMSO and 1/10 volumeof a commercial reagent (GC-Melt™; Clontech Laboratories, Inc.). Thereaction was run for 1 minute at 94 degrees; then 30 cycles of 94degrees, 20 seconds; 67 degrees, 1 minute; then a final 5-minuteincubation at 72 degrees. A resulting 833-bp product was sequenced andfound to be a zvegf4 fragment containing the remainder of the codingsequence with an intiation MET codon, upstream stop codon, and 5′untranslated sequence. The composite sequence included an open readingframe of 1,110 bp (SEQ ID NO:1).

Example 3

To make transgenic animals expressing zvegf4 genes requires adult,fertile males (studs) (B6C3f1, 2-8 months of age (Taconic Farms,Germantown, N.Y.)), vasectomized males (duds) (B6D2f1, 2-8 months,(Taconic Farms)), prepubescent fertile females (donors) (B6C3f1, 4-5weeks, (Taconic Farms)) and adult fertile females (recipients) (B6D2f1,2-4 months, (Taconic Farms)).

The donors are acclimated for 1 week, then injected with approximately 8IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma, St. Louis, Mo.)I.P., and 46-47 hours later, 8 IU/mouse of human Chorionic Gonadotropin(hCG (Sigma)) I.P. to induce superovulation. Donors are mated with studssubsequent to hormone injections. Ovulation generally occurs within 13hours of hCG injection. Copulation is confirmed by the presence of avaginal plug the morning following mating.

Fertilized eggs are collected under a surgical scope (Leica MZ12 StereoMicroscope; Leica, Wetzlar, Germany). The oviducts are collected andeggs are released into urinanalysis slides containing hyaluronidase(Sigma Chemical Co.). Eggs are washed once in hyaluronidase, and twicein Whitten's W640 medium (Table 7; all reagents available from SigmaChemical Co.) that has been incubated with 5% CO₂, 5% O₂, and 90% N₂ at37° C. The eggs are stored in a 37° C./5% CO₂ incubator untilmicroinjection.

TABLE 7 mgs/200 ml mgs/500 ml NaCl 1280 3200 KCl 72 180 KH₂PO₄ 32 80MgSO₄•7H₂O 60 150 Glucose 200 500 Ca²⁺ Lactate 106 265 Benzylpenicillin15 37.5 Streptomycin SO₄ 10 25 NaHCO₃ 380 950 Na Pyruvate 5 12.5 H₂0 200 ml  500 ml 500 mM EDTA 100 μl 250 μl 5% Phenol Red 200 μl 500 μlBSA 600 1500

Zvegf4 cDNA is inserted into the expression vector pHB12-8 (see FIG. 2).Vector pHB12-8 was derived from p2999B4 (Palmiter et al., Mol. Cell.Biol. 13:5266-5275, 1993) by insertion of a rat insulin II intron (ca.200 bp) and polylinker (Fse I/Pme I/Asc I) into the Nru I site. Thevector comprises a mouse metallothionein (MT-1) promoter (ca. 750 bp)and human growth hormone (hGH) untranslated region and polyadenylationsignal (ca. 650 bp) flanked by 10 kb of MT-1 5′ flanking sequence and 7kb of MT-1 3′ flanking sequence. The cDNA is inserted between theinsulin II and hGH sequences.

10-20 micrograms of plasmid DNA is linearized, gel-purified, andresuspended in 10 mM Tris pH 7.4, 0.25 mM EDTA pH 8.0, at a finalconcentration of 5-10 nanograms per microliter for microinjection.

Plasmid DNA is microinjected into harvested eggs contained in a drop ofW640 medium overlaid by warm, CO₂-equilibrated mineral oil. The DNA isdrawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary) and injected into individual eggs. Eachegg is penetrated with the injection needle into one or both of thehaploid pronuclei.

Picoliters of DNA are injected into the pronuclei, and the injectionneedle is withdrawn without coming into contact with the nucleoli. Theprocedure is repeated until all the eggs are injected. Successfullymicroinjected eggs are transferred into an organ tissue-culture dishwith pregassed W640 medium for storage overnight in a 37° C./5% CO₂incubator.

The following day, 2-cell embryos are transferred into pseudopregnantrecipients. The recipients are identified by the presence of copulationplugs, after copulating with vasectomized duds. Recipients areanesthetized and shaved on the dorsal left side and transferred to asurgical microscope. A small incision is made in the skin and throughthe muscle wall in the middle of the abdominal area outlined by theribcage, the saddle, and the hind leg, midway between knee and spleen.The reproductive organs are exteriorized onto a small surgical drape.The fat pad is stretched out over the surgical drape, and a babyserrefine (Roboz, Rockville, Md.) is attached to the fat pad and lefthanging over the back of the mouse, preventing the organs from slidingback in.

With a fine transfer pipette containing mineral oil followed byalternating W640 and air bubbles, 12-17 healthy 2-cell embryos from theprevious day's injection are transferred into the recipient. The swollenampulla is located, and, holding the oviduct between the ampulla and thebursa, a nick in the oviduct is made with a 28 g needle close to thebursa, making sure not to tear the ampulla or the bursa.

The pipette is transferred into the nick in the oviduct, and the embryosare blown in, allowing the first air bubble to escape the pipette. Thefat pad is gently pushed into the peritoneum, and the reproductiveorgans are allowed to slide in. The peritoneal wall is closed with onesuture, and the skin is closed with a wound clip. The mice recuperate ona 37° C. slide warmer for a minimum of 4 hours.

The recipients are returned to cages in pairs, and allowed 19-21 daysgestation. After birth, 19-21 days postpartum is allowed before weaning.The weanlings are sexed and placed into separate sex cages, and a 0.5 cmbiopsy (used for genotyping) is snipped off the tail with cleanscissors.

Genomic DNA is prepared from the tail snips using a commerciallyavailable kit (DNeasy™ 96 Tissue Kit; Qiagen, Valencia, Calif.)following the manufacturer's instructions. Genomic DNA is analyzed byPCR using primers designed to the human growth hormone (hGH) 3′ UTRportion of the transgenic vector. The use of a region unique to thehuman sequence (identified from an alignment of the human and mousegrowth hormone 3′ UTR DNA sequences) ensures that the PCR reaction doesnot amplify the mouse sequence. Primers ZC17,251 (SEQ ID NO:28) andZC17,252 (SEQ ID NO:29) amplify a 368-base-pair fragment of hGH. Inaddition, primers ZC17,156 (SEQ ID NO:30) and ZC17,157 (SEQ ID NO:31),which hybridize to vector sequences and amplify the cDNA insert, may beused along with the hGH primers. In these experiments, DNA from animalspositive for the transgene will generate two bands, a 368-base-pair bandcorresponding to the hGH 3′ UTR fragment and a band of variable sizecorresponding to the cDNA insert.

Once animals are confirmed to be transgenic (TG), they are back-crossedinto an inbred strain by placing a TG female with a wild-type male, or aTG male with one or two wild-type female(s). As pups are born andweaned, the sexes are separated, and their tails snipped for genotyping.

To check for expression of a transgene in a live animal, a partialhepatectomy is performed. A surgical prep is made of the upper abdomendirectly below the xiphoid process. Using sterile technique, a small1.5-2 cm incision is made below the sternum, and the left lateral lobeof the liver is exteriorized. Using 4-0 silk, a tie is made around thelower lobe securing it outside the body cavity. An atraumatic clamp isused to hold the tie while a second loop of absorbable Dexon (AmericanCyanamid, Wayne, N.J.) is placed proximal to the first tie. A distal cutis made from the Dexon tie, and approximately 100 mg of the excisedliver tissue is placed in a sterile petri dish. The excised liversection is transferred to a 14-ml polypropylene round bottom tube, snapfrozen in liquid nitrogen, and stored on dry ice. The surgical site isclosed with suture and wound clips, and the animal's cage is placed on a37° C. heating pad for 24 hours post-operatively. The animal is checkeddaily post-operatively, and the wound clips are removed 7-10 days aftersurgery.

Analysis of the mRNA expression level of each transgene is done using anRNA solution hybridization assay or real-time PCR on an ABI Prism 7700(PE Applied Biosystems, Inc., Foster City, Calif.) following themanufacturer's instructions.

Example 4

An expression plasmid containing all or part of a polynucleotideencoding zvegf4 is constructed via homologous recombination. A fragmentof zvegf4 cDNA is isolated by PCR using the polynucleotide sequence ofSEQ ID NO: 1 with flanking regions at the 5′ and 3′ ends correspondingto the vector sequences flanking the zvegf4 insertion point. The primersfor PCR each include from 5′ to 3′ end: 40 bp of flanking sequence fromthe vector and 17 bp corresponding to the amino and carboxyl terminifrom the open reading frame of zvegf4.

Ten μl of the 100 μl PCR reaction is run on a 0.8% LMP agarose gel(Seaplaque GTG) with 1×TBE buffer for analysis. The remaining 90 μl ofPCR reaction is precipitated with the addition of 5 μl 1 M NaCl and 250μl of absolute ethanol. The plasmid pZMP6, which has been cut with SmaI,is used for recombination with the PCR fragment. Plasmid pZMP6 wasconstructed from pZP9 (deposited at the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, underAccession No. 98668) with the yeast genetic elements taken from pRS316(deposited at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, under Accession No. 77145), aninternal ribosome entry site (IRES) element from poliovirus, and theextracellular domain of CD8 truncated at the C-terminal end of thetransmembrane domain. pZMP6 is a mammalian expression vector containingan expression cassette having the mouse metallothionein-1 promoter,multiple restriction sites for insertion of coding sequences, a stopcodon, and a human growth hormone terminator. The plasmid also containsan E. coli origin of replication; a mammalian selectable markerexpression unit comprising an SV40 promoter, enhancer and origin ofreplication, a DHFR gene, and the SV40 terminator; as well as the URA3and CEN-ARS sequences required for selection and replication in S.cerevisiae.

One hundred microliters of competent yeast cells (S. cerevisiae) areindependently combined with 10 μl of the various DNA mixtures from aboveand transferred to a 0.2-cm electroporation cuvette. The yeast/DNAmixtures are electropulsed at 0.75 kV (5 kV/cm), ∞ ohms, 25 μF. To eachcuvette is added 600 μl of 1.2 M sorbitol, and the yeast is plated intwo 300-μl aliquots onto two URA-D plates and incubated at 30° C. Afterabout 48 hours, the Ura yeast transformants from a single plate areresuspended in 1 ml H₂O and spun briefly to pellet the yeast cells. Thecell pellet is resuspended in 1 ml of lysis buffer (2% Triton X-100, 1%SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture is added to an Eppendorf tubecontaining 300 μl acid-washed glass beads and 200 phenol-chloroform,vortexed for 1 minute intervals two or three times, and spun for 5minutes in an Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase is transferred to a fresh tube, and theDNA is precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet is resuspended in10 μl H₂O.

Transformation of electrocompetent E. coli host cells (Electromax DH10B™cells; obtained from Life Technologies, Inc., Gaithersburg, Md.) is donewith 0.5-2 ml yeast DNA prep and 40 μl of cells. The cells areelectropulsed at 1.7 kV, 25 and 400 ohms. Following electroporation, 1ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) is plated in 250-μl aliquots on four LB AMP plates (LB broth(Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/L Ampicillin).

Individual clones harboring the correct expression construct for zvegf4are identified by restriction digest to verify the presence of thezvegf4 insert and to confirm that the various DNA sequences have beenjoined correctly to one another. The inserts of positive clones aresubjected to sequence analysis. Larger scale plasmid DNA is isolatedusing a commercially available kit (QIAGEN Plasmid Maxi Kit, Qiagen,Valencia, Calif.) according to manufacturer's instructions. The correctconstruct is designated zvegf4/pZMP6.

Example 5

CHO DG44 cells (Chasin et al., Som. Cell. Molec. Genet. 12:555-566,1986) are plated in 10-cm tissue culture dishes and allowed to grow toapproximately 50% to 70% confluency overnight at 37° C., 5% CO₂, inHam's F12/FBS media (Ham's F12 medium, Life Technologies), 5% fetalbovine serum (Hyclone, Logan, Utah), 1% L-glutamine (JRH Biosciences,Lenexa, Kans.), 1% sodium pyruvate (Life Technologies). The cells arethen transfected with the plasmid zvegf4/pZMP6 by liposome-mediatedtransfection using a 3:1 (w/w) liposome formulation of the polycationiclipid2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium-trifluoroacetateand the neutral lipid dioleoyl phosphatidylethanolamine inmembrane-filtered water (Lipofectamine™ Reagent, Life Technologies), inserum free (SF) media formulation (Ham's F12, 10 mg/ml transferrin, 5mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).Zvegf4/pZMP6 is diluted into 15-ml tubes to a total final volume of 640μl with SF media. 35 μl of Lipofectamine™ is mixed with 605 μl of SFmedium. The Lipofectamine™ mixture is added to the DNA mixture andallowed to incubate approximately 30 minutes at room temperature. Fiveml of SF media is added to the DNA:Lipofectamine™ mixture. The cells arerinsed once with 5 ml of SF media, aspirated, and the DNA:Lipofectamine™mixture is added. The cells are incubated at 37° C. for five hours, then6.4 ml of Ham's F12/10% FBS, 1% PSN media is added to each plate. Theplates are incubated at 37° C. overnight, and the DNA:Lipofectamine™mixture is replaced with fresh 5% FBS/Ham's media the next day. On day 3post-transfection, the cells are split into T-175 flasks in growthmedium. On day 7 post-transfection, the cells are stained withFITC-anti-CD8 monoclonal antibody (Pharmingen, San Diego, Calif.)followed by anti-FITC-conjugated magnetic beads (Miltenyi Biotec,Auburn, Calif.). The CD8-positive cells are separated using commerciallyavailable columns (MiniMACS Separation Unit; Miltenyi Biotec) accordingto the manufacturer's directions and put into DMEM/Ham's F12/5% FBSwithout nucleosides but with 50 nM methotrexate (selection medium).

Cells are plated for subcloning at a density of 0.5, 1 and 5 cells perwell in 96-well dishes in selection medium and allowed to grow out forapproximately two weeks. The wells are checked for evaporation of mediumand brought back to 200 μl per well as necessary during this process.When a large percentage of the colonies in the plate are nearconfluency, 100 μl of medium is collected from each well for analysis bydot blot, and the cells are fed with fresh selection medium. Thesupernatant is applied to a nitrocellulose filter in a dot blotapparatus, and the filter is treated at 100° C. in a vacuum oven todenature the protein. The filter is incubated in 625 mM Tris-glycine, pH9.1, 5 mM β-mercaptoethanol, at 65° C., 10 minutes, then in 2.5% non-fatdry milk Western A Buffer (0.25% gelatin, 50 mM Tris-HCl pH 7.4, 150 mMNaCl, 5 mM EDTA, 0.05% Igepal CA-630) overnight at 4° C. on a rotatingshaker. The filter is incubated with the anti-CD8 antibody-HRP conjugatein 2.5% non-fat dry milk Western A buffer for 1 hour at room temperatureon a rotating shaker. The filter is then washed three times at roomtemperature in PBS plus 0.01% Tween 20, 15 minutes per wash. The filteris developed with chemiluminescence reagents (ECL™ direct labeling kit;Amersham Corp., Arlington Heights, Ill.) according to the manufacturer'sdirections and exposed to film (Hyperfilm ECL, Amersham) forapproximately 5 minutes. Positive clones are trypsinized from the96-well dish and transferred to E-well dishes in selection medium forscaleup and analysis by Western blot.

Example 6

The protein coding region of zvegf4 is amplified by PCR using primersthat add FseI and AscI restriction sites at the 5′ and 3′ termini,respectively. PCR primers are used with a template containing thefull-length zvegf4 cDNA in a PCR reaction as follows: one cycle at 95°C. for 5 minutes; followed by 15 cycles at 95° C. for 1 min., 58° C. for1 min., and 72° C. for 1.5 min.; followed by 72° C. for 7 min.; followedby a 4° C. soak. The PCR reaction product is loaded onto a 1.2% (lowmelt) (SeaPlaque GTG™; FMC, Rockland, Me.) gel in TAE buffer. The zvegf4PCR product is excised from the gel and purified using a spin columncontaining a silica gel membrane (QIAquick™ Gel Extraction Kit; Qiagen,Inc., Valencia, Calif.) as per kit instructions. The PCR product is thendigested, phenol/chloroform extracted, EtOH precipitated, and rehydratedin 20 ml TE (Tris/EDTA pH 8). The zvegf4 fragment is then ligated intothe cloning sites of the transgenic vector pHB12-8 and transformed intoE. coli host cells (Electromax DH10B™ cells; obtained from LifeTechnologies, Inc., Gaithersburg, Md.) by electroporation. Clonescontaining zvegf4 DNA are identified by restriction analysis. A positiveclone is confirmed by direct sequencing.

The zvegf4 cDNA is released from the pTG12-8 vector using FseI and AscIenzymes. The cDNA is isolated on a 1% low melt agarose gel, and is thenexcised from the gel. The gel slice is melted at 70° C., extracted twicewith an equal volume of Tris buffered phenol, and EtOH precipitated. TheDNA is resuspended in 10 μl H₂O.

The zvegf4 cDNA is cloned into the FseI-AscI sites of a modifiedpAdTrack CMV (He et al., Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998).This construct contains a GFP marker gene. The CMV promoter driving GBPexpression has been replaced with the SV40 promoter, and the SV40polyadenylation signal has been replaced with the human growth hormonepolyadenylation signal. In addition, the native polylinker has beenreplaced with FseI, EcoRV, and AscI sites. This modified form ofpAdTrack CMV was named pZyTrack. Ligation is performed using a DNAligation and screening kit (Fast-Link™; Epicentre Technologies, Madison,Wis.). In order to linearize the plasmid, approximately 5 μg of thepZyTrack zvegf4 plasmid is digested with PmeI. Approximately 1 μg of thelinearized plasmid is cotransformed with 200 ng of supercoiled pAdEasy(He et al., ibid.) into BJ5183 cells. The co-transformation is doneusing a Bio-Rad Gene Pulser at 2.5 kV, 200 ohms and 25 μf. The entireco-transformation is plated on 4 LB plates containing 25 μg/mlkanamycin. The smallest colonies are picked and expanded inLB/kanamycin, and recombinant adenovirus DNA identified by standard DNAminiprep procedures. Digestion of the recombinant adenovirus DNA withFseI-AscI confirms the presence of zvegf4 DNA. The recombinantadenovirus miniprep DNA is transformed into E. coli DH10B competentcells, and DNA is prepared therefrom.

Approximately 5 μg of recombinant adenoviral DNA is digested with PacIenzyme (New England Biolabs) for 3 hours at 37° C. in a reaction volumeof 100 μl containing 20-30 U of PacI. The digested DNA is extractedtwice with an equal volume of phenol/chloroform and precipitated withethanol. The DNA pellet is resuspended in 10 μl distilled water. A T25flask of QBI-293A cells (Quantum Biotechnologies, Inc., Montreal,Canada), inoculated the day before and grown to 60-70% confluence, aretransfected with the Pad digested DNA. The PacI-digested DNA is dilutedup to a total volume of 50 μl with sterile HBS (150 mM NaCl, 20 mMHEPES). In a separate tube, 20 μl of 1 mg/mlN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate(DOTAP; Boehringer Mannheim) is diluted to a total volume of 100 μl withHBS. The DNA is added to the DOTAP, mixed gently by pipeting up anddown, and left at room temperature for 15 minutes. The media is removedfrom the 293A cells and washed with 5 ml serum-free MEM-alpha (LifeTechnologies, Gaithersburg, Md.) containing 1 mM sodium pyruvate (LifeTechnologies), 0.1 mM MEM non-essential amino acids (Life Technologies)and 25 mM HEPES buffer (Life Technologies). 5 ml of serum-free MEM isadded to the 293A cells and held at 37° C. The DNA/lipid mixture isadded drop-wise to the T25 flask of 293A cells, mixed gently, andincubated at 37° C. for 4 hours. After 4 hours the media containing theDNA/lipid mixture is aspirated off and replaced with 5 ml complete MEMcontaining 5% fetal bovine serum. The transfected cells are monitoredfor Green Fluorescent Protein (GFP) expression and formation of foci(viral plaques).

Seven days after transfection of 293A cells with the recombinantadenoviral DNA, the cells expressing the GFP protein start to form foci.These foci are viral “plaques” and the crude viral lysate is collectedby using a cell scraper to collect all of the 293A cells. The lysate istransferred to a 50 ml conical tube. To release most of the virusparticles from the cells, three freeze/thaw cycles are done in a dryice/ethanol bath and a 37° C. waterbath.

Ten 10-cm plates of nearly confluent (80-90%) 293A cells are set up 20hours prior to infection. The crude lysate is amplified (primaryamplification) to obtain a working “stock” of zvegf4 rAdV lysate. 200 mlof crude rAdV lysate is added to each 10-cm plate, and the plates aremonitored for 48 to 72 hours looking for cytopathic effect (CPE) underthe white light microscope and expression of GIP under the fluorescentmicroscope. When all of the 293A cells show CPE, this 1° stock lysate iscollected, and freeze/thaw cycles performed as described above.

Secondary (2°) amplification of zvegf4 rAdV is obtained from twenty15-cm tissue culture dishes of 80-90% confluent 293A cells. All but 20ml of 5% MEM media is removed, and each dish is inoculated with 300-500ml of 1° amplified rAdv lysate. After 48 hours the 293A cells are lysedfrom virus production, the lysate is collected into 250 ml polypropylenecentrifuge bottles, and the rAdV is purified.

NP-40 detergent is added to a final concentration of 0.5% to the bottlesof crude lysate to lyse all cells. Bottles are placed on a rotatingplatform for 10 minutes and agitated as fast as possible. The debris ispelleted by centrifugation at 20,000×G for 15 minutes. The supernatantis transferred to 250-ml polycarbonate centrifuge bottles, and 0.5volume of 20% PEG8000/2.5M NaCl solution is added. The bottles areshaken overnight on ice. The bottles are centrifuged at 20,000×G for 15minutes, and the supernatants are discarded into a bleach solution. Awhite precipitate (precipitated virus/PEG) forms in two vertical linesalong the walls of the bottles on either side of the spin mark. Using asterile cell scraper, the precipitate from 2 bottles is resuspended in2.5 ml PBS. The virus solution is placed in 2-ml microcentrifuge tubesand centrifuged at 14,000×G in a microcentrifuge for 10 minutes toremove any additional cell debris. The supernatants from the 2-mlmicrocentrifuge tubes are transferred into a 15-ml polypropylene snapcaptube and adjusted to a density of 1.34 g/ml with CsCl. The volume of thevirus solution is estimated, and 0.55 g/ml of CsCl added. The CsCl isdissolved, and 1 ml of this solution weighed. The solution istransferred to polycarbonate, thick-walled, 3.2 ml centrifuge tubes(Beckman) and spun at 348,000 X G for 3-4 hours at 25° C. The virusforms a white band. Using wide-bore pipette tips, the virus band iscollected.

The virus from the gradient will have a large amount of CsCl, which mustbe removed before it can be used on cells. Pharmacia PD-10 columnsprepacked with Sephadex® G-25M (Pharmacia) are used to desalt the viruspreparation. The column is equilibrated with 20 ml of PBS. The virus isloaded and allowed to run into the column. 5 ml of PBS is added to thecolumn, and fractions of 8-10 drops collected. The optical density of1:50 dilutions of each fraction is determined at 260 nm on aspectrophotometer, and a clear absorbance peak is identified. Thesefractions are pooled, and the optical density (OD) of a 1:25 dilution isdetermined. OD is converted into virus concentration using the formula(OD at 260 nm)(25)(1.1×10¹²)=virions/ml.

To store the virus, glycerol is added to the purified virus to a finalconcentration of 15%, mixed gently and stored in aliquots at −80° C.

A protocol developed by Quantum Biotechnologies, Inc. (Montreal, Canada)is followed to measure recombinant virus infectivity. Briefly, two96-well tissue culture plates are seeded with 1×10⁴ 293A cells per wellin MEM containing 2% fetal bovine serum for each recombinant virus to beassayed. After 24 hours, 10-fold dilutions of each virus from 1×10⁻² to1×10⁻¹⁴ are made in MEM containing 2% fetal bovine serum. 100 μl of eachdilution is placed in each of 20 wells. After 5 days at 37° C., wellsare read either positive or negative for CPE and PFU/ml is calculated.

TCID₅₀ formulation used is as per Quantum Biotechnologies, Inc., above.The titer (T) is determined from a plate where virus used is dilutedfrom 10² to 10¹⁴, and read 5 days after the infection. At each dilutiona ratio (R) of positive wells for CPE per the total number of wells isdetermined. The titer of the undiluted sample is T=10^((1+F))=TCID₅₀/ml,where F=1+d(S−0.5), S is the sum of the ratios (R), and d is Log₁₀ ofthe dilution series (e.g., d=1 for a ten-fold dilution series). Toconvert TCID₅₀/ml to pfu/ml, 0.7 is subtracted from the exponent in thecalculation for titer (T).

Example 7

Recombinant zvegf4 having a carboxyl-terminal Glu-Glu affinity tag wasproduced in a baculovirus expression system according to conventionalmethods. The culture was harvested, and the cells were lysed with asolution of 0.02 M Tris-HCl, pH 8.3, 1 mM EDTA, 1 mM DTT, 1 mM4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride (Pefabloc® SC;Boehringer-Mannheim), 0.5 μM aprotinin, 4 mM leupeptin, 4 mM E-64, 1%NP-40 at 4° C. for 15 minutes on a rotator. The solution wascentrifuged, and the supernatant was recovered. Twenty ml of extract wascombined with 50 μl of anti-Glu-Glu antibody conjugated to Sepharose®beads in 50 μl buffer. The mixture was incubated on a rotator at 4° C.overnight. The beads were recovered by centrifugation and washed 3×15minutes at 4° C. Pellets were combined with sample buffer containingreducing agent and heated at 98° C. for five minutes. The protein wasanalyzed by polyacrylamide gel electrophoresis under reducing conditionsfollowed by western blotting on a PVDF membrane using an antibody to theaffinity tag. Two bands were detected, one a M_(r)=49 kD and the otherat M_(r)=21 kD. Sequence analysis showed the larger band to comprise twosequences, one beginning at Arg-19 of SEQ ID NO:2 and the otherbeginning at Asn-35 of SEQ ID NO:2. The asparagine residue appeared tohave been deamidated to an aspartic acid. The smaller band began atSer-250 of SEQ ID NO:2.

Example 8

The zvegf4 cDNA was cloned into the EcoRV-AscI sites of a modifiedpAdTrack-CMV (He et al., Proc. Natl. Acad. Sci. USA 95:2509-2514, 1998).This construct contains the green fluorescent protein (GFP) marker gene.The CMV promoter driving GFP expression was replaced with the SV40promoter, and the SV40 polyadenylation signal was replaced with thehuman growth hormone polyadenylation signal. In addition, the nativepolylinker was replaced with FseI, EcoRV, and AscI sites. This modifiedform of pAdTrack-CMV was named pZyTrack. Ligation was performed using acommercially available DNA ligation and screening kit (Fast-Link™ kit;Epicentre Technologies, Madison, Wis.).

Zvegf4 was assayed in an aortic ring outgrowth assay (Nicosia andOttinetti, ibid.; Villaschi and Nicosia, ibid.). Thoracic aortas wereisolated from 1-2 month old SD male rats and transferred to petri dishescontaining HANK's buffered salt solution. The aortas were flushed withadditional HANK's buffered salt solution to remove blood, andadventitial tissue surrounding the aorta was carefully removed. Cleanedaortas were transferred to petri dishes containing EBM basal media,serum free (Clonetics, San Diego, Calif.). Aortic rings were obtained byslicing approximately 1-mm sections using a scalpel blade. The ends ofthe aortas used to hold the aorta in place were not used. The rings wererinsed in fresh EBM basal media and placed individually in a wells of a24-well plate coated with basement membrane matrix (Matrigel®; BectonDickinson, Franklin Lakes, N.J.). The rings were overlayed with anadditional 50 of the matrix solution and placed at 37° C. for 30 minutesto allow the matrix to gel. Test samples were diluted in EBM basalserum-free media supplemented with 100 units/ml penicillin, 100 μg/mlstreptomycin and HEPES buffer and added at 1 ml/well. Background controlwas EBM basal serum-free media alone. Basic FGF (R&D Systems,Minneapolis, Minn.) at 20 ng/ml was used as a positive control. Zvegf4adenovirus was added to wells, assuming a cell count of 500,000 cellsand a multiplicity of infection of 5000 particles/cell. A nulladenovirus (designated “zPar”) was used as a control. Samples were addedin a minimum of quadruplets. Rings were incubated for 5-7 days at 37° C.and analyzed for growth. Aortic outgrowth was scored by multiple,blinded observers using 0 as no growth and 4 as maximum growth. Zvegf4adenovirus produced a significant increase in outgrowth, comparable tothe most potent control (bFGF).

Example 9

Polyclonal anti-peptide antibodies were prepared by immunizing 2 femaleNew Zealand white rabbits with the peptides huzvegf4-1(CGHKEVPPRIKSRTNQIK; SEQ ID NO:39), huzvegf4-2(ESWQEDLENMYLDTPRYRGRSYHDC; SEQ ID NO:40), or huzvegf4-3(CFEPGHIKRRGRAKTMALVDIQLD; SEQ ID NO:41). The peptides were synthesizedusing an Applied Biosystems Model 431A peptide synthesizer (AppliedBiosystems, Inc., Foster City, Calif.) according to the manufacturer'sinstructions. The peptides were conjugated to keyhole limpet hemocyanin(KLH) with maleimide activation. The rabbits were each given an initialintraperitoneal (ip) injection of 200 μg of peptide in Complete Freund'sAdjuvant followed by booster ip injections of 100 μg peptide inIncomplete Freund's Adjuvant every three weeks. Seven to ten days afterthe administration of the second booster injection (3 total injections),the animals were bled, and the sea were collected. The animals were thenboosted and bled every three weeks.

The zvegf4 peptide-specific rabbit sera were characterized by an ELISAtiter check using 1 μg/ml of the peptide used to make the antibody as anantibody target. The 2 rabbit sera to the huzvegf4-1 peptide had titerto their specific peptide at a dilution of 1:5,000,000. The 2 rabbitsera to the huzvegf4-2 peptide had titer to their specific peptide at adilution of 1:5,000,000. The 2 rabbit seras to the huzvegf4-3 peptidehad titer to their specific peptide at a dilution of 1:500,000.

The zvegf4 peptide-specific polyclonal antibodies were affinity purifiedfrom the sera using CNBr-SEPHAROSE 4B protein columns (Pharmacia LKB)that were prepared using 10 mg of the specific peptide per gramCNBr-SEPHAROSE, followed by 20× dialysis in PBS overnight.Zvegf4-specific antibodies were characterized by an ELISA titer checkusing 1 μg/ml of the appropriate peptide antigens as antibody targets.The lower limit of detection (LLD) of the anti-huzvegf4-1 affinitypurified antibody on its specific antigen (huzvegf4-1 peptide) was adilution of 0.1 pg/ml. The LLD of the anti-huzvegf4-2 affinity purifiedantibody on its specific antigen (huzveg4-2 peptide) was a dilution of 5ng/ml. The LLD of the rabbit anti-huzvegf4-3 affinity purified antibodyon its specific antigen (huzvegf4-3 peptide) was a dilution of 5 ng/ml.

Example 10

Recombinant carboxyl-terminal Glu-Glu tagged zvegf4 (zvegf4-cee) wasproduced from recombinant baculovirus-infected insect cells. Two-litercultures were harvested, and the media were sterile-filtered using a 0.2μm filter.

Protein was purified from the conditioned media by a combination ofanti-Glu-Glu (anti-EE) peptide antibody affinity chromatography andS-200 gel exclusion chromatography. Culture media (pH 6.0, conductivity7 mS) was directly loaded onto a 20×80 mm (25-ml bed volume) anti-EEantibody affinity column at a flow of 6 ml/minute. The column was washedwith ten column volumes of PBS, then bound protein was eluted with twocolumn volumes of 0.4 mg/ml EYMPTD peptide (SEQ ID NO:42) (PrincetonBioMolecules Corp., Langhorne, Pa.). Five-ml fractions were collected.Samples from the anti-EE antibody affinity column were analyzed bySDS-PAGE with silver staining and western blotting (as disclosed below)for the presence of zvegf4-cee. Zvefg4-cee-containing fractions werepooled and concentrated to 3.8 ml by filtration using a Biomax™-5concentrator (Millipore Corp., Bedford, Mass.), and loaded onto a16×1000 mm gel filtration column (Sephacryl™ S-200 HR; AmershamPharmacia Biotech, Piscataway, N.J.). The fractions containing purifiedzvegf4-cee were pooled, filtered through a 0.2 μm filter, aliquoted into100 μl each, and frozen at −80° C. The concentration of the finalpurified protein was determined by colorimetric assay (BCA assayreagents; Pierce, Rockford, Ill.) and HPLC-amino acid analysis.

Recombinant zvegf4-cee was analyzed by SDS-PAGE (NuPAGE™ 4-12% gel;Novex, San Diego, Calif.) with silver staining (FASTsilver™, GenoTechnology, Inc., Maplewood, Mo.) and Western blotting using antibodiesto the huzvegf4-1, huzvegf4-2, and huzvefg4-3 peptides, and anti-EEantibody. Either the conditioned media or purified protein waselectrophoresed using an electrophoresis mini-cell (XCell II™ mini-cell;Novex, San Diego, Calif.) and transferred to nitrocellulose (0.2 lam;Bio-Rad Laboratories, Hercules, Calif.) at room temperature using anXCell II™ blot module (Novex) with stirring according to directionsprovided in the instrument manual. The transfer was run at 500 mA forone hour in a buffer containing 25 mM Tris base, 200 mM glycine, and 20%methanol. The filters were then blocked with 10% non-fat dry milk in PBSfor 10 minutes at room temperature. The nitrocellulose was quicklyrinsed, then primary antibody was added in PBS containing 2.5% non-fatdry milk. The blots were incubated for two hours at room temperature orovernight at 4° C. with gentle shaking. Following the incubation, blotswere washed three times for 10 minutes each in PBS. Secondary antibody(goat anti-rabbit IgG conjugated to horseradish peroxidase; obtainedfrom Rockland Inc., Gilbertsville, Pa.) diluted 1:2000 in PBS containing2.5% non-fat dry milk was added, and the blots were incubated for twohours at room temperature with gentle shaking. The blots were thenwashed three times, 10 minutes each, in PBS, then quickly rinsed in H₂O.The blots were developed using commercially available chemiluminescentsubstrate reagents (SuperSignal® ULTRA reagents 1 and 2 mixed 1:1;reagents obtained from Pierce), and the signal was captured using imageanalysis software (Lumi-Imager™ Lumi Analyst 3.0; Roche MolecularBiochemicals, Indianapolis, Ind.) for times ranging from 10 seconds to 5minutes or as necessary.

The purified zvefg4-cee appeared as a single band at about 85 kDa undernon-reducing conditions with silver staining, but at about 50 kDa underreducing conditions, suggesting a dimeric form of zvefg4-cee undernon-reducing conditions.

Using either 4-1, 4-3 or anti-EE antibody, the purified zvegf4-ceeshowed the same result as silver staining gel; the 4-3 antibody gave amuch weaker signal. However, in addition to recognizing the 85-kDa bandunder non-reducing conditions and the 50-kDa band under reducingconditions, the 4-2 antibody recognized two bands at 35 kDa and 32 kDaunder non-reducing conditions, and two bands at 38 kDa and 35 kDa underreducing conditions. While not wishing to be bound by theory, thesmaller bands are likely to be cleaved forms of zvefg4-cee missing theN-terminal portion of the protein that is recognized by the 4-1antibody.

Example 11

Recombinant zvegf4 was analyzed for mitogenic activity on rat liverstellate cells (obtained from N. Fausto, University of Washington),human aortic smooth muscle cells (Clonetics Corp., Walkersville, Md.),human retinal pericytes (Clonetics Corp.) and human hepatic fibroblasts(Clonetics Corp.). Test samples consisted of conditioned media (CM) fromadenovirally infected HaCaT human keratinocyte cells (Boukamp et al., J.Cell. Biol. 106:761-771, 1988; Skobe and Fusenig, Proc. Natl. Acad. Sci.USA 95:1050-1055, 1998; obtained from Dr. Norbert E. Fusenig, DeutschesKrebsforschungszentrum, Heidelberg, Germany) expressing full lengthzvegf-4. Control CM was generated from HaCaT cells infected with aparental GFP-expressing adenovirus (zPar). The CM were concentrated10-fold using a 15 ml centrifugal filter device with a 10K membranefilter (Ultrafree®; Millipore Corp., Bedford, Mass.), then diluted backto 1× with ITS media (serum-free DMEM/Ham's F-12 medium containing 5μg/ml insulin, 20 μg/ml transferrin, and 16 μg/ml selenium). Cells wereplated at a density of 2,000 cells/well in 96-well culture plates andgrown for approximately 72 hours in DMEM containing 10% fetal calf serumat 37° C. Cells were quiesced by incubating them for approximately 20hours in serum-free DMEM/Ham's F-12 medium containing insulin (5 μg/ml),transferrin (20 μg/ml), and selenium (16 pg/ml) (ITS). At the time ofthe assay, the medium was removed, and test samples were added to thewells in triplicate. For measurement of [³H]thymidine incorporation, 20μl of a 50 μCi/ml stock in DMEM was added directly to the cells, for afinal activity of 1 μCi/well. After another 24-hour incubation, mediawere removed and cells were incubated with 0.1 ml of trypsin until cellsdetached. Cells were harvested onto 96-well filter plates using a sampleharvester (FilterMate™ harvester; Packard Instrument Co., Meriden,Conn.). The plates were then dried at 65° C. for 15 minutes, sealedafter adding 40 μl/well scintillation cocktail (Microscint™ 0; PackardInstrument Co.) and counted on a microplate scintillation counter(Topcount®; Packard Instrument Co.). Results, presented in Table 8,demonstrated that zvegf4 CM had approximately 7-fold higher mitogenicactivity than control CM on pericytes cells and approximately a1.5-2.4-fold higher mitogenic activity on the other cell types tested.

TABLE 8 CPM incorporated Zvegf4 (1x CM) zPar (1xCM) Sample Mean St. dev.Mean St. dev. Human retinal pericytes 3621 223 523 306 Human hepaticfibroblasts 7757 753 3232 264 Human aortic SMC 2009 37 1263 51 Rat liverstellate cells 34707 1411 14413 1939

Example 12

Recombinant, C terminal glu-glu tagged zvegf4 was analyzed for mitogenicactivity on human aortic smooth muscle cells (HAoSMC) (Clonetics), humanretinal pericytes (Clonetics) and human aortic adventitial fibroblasts(AoAF) (Clonetics). Cells were plated at a density of 2,000 cells/wellin 96-well culture plates and grown for approximately 72 hours in DMEMcontaining 10% fetal calf serum at 37° C. Cells were quiesced byincubating them for 20 hours in ITS medium. At the time of the assay,the medium was removed, and test samples were added to the wells intriplicate. Test samples consisted of purified, full-length, taggedzvegf4 expressed in baculovirus-infected cells. Purified protein in abuffer containing 0.1% BSA was serially diluted into ITS medium atconcentrations of 1 μg/ml to 1 ng/ml and added to the test plate. Acontrol buffer of 0.1% BSA was diluted identically to the highestconcentration of zvegf4 protein and added to the plate. For measurementof [³H]thymidine incorporation, 20 μl of a 50 μCi/ml stock in DMEM wasadded directly to the cells, for a final activity of 1 μCi/well. Afteranother 24-hour incubation, mitogenic activity was assessed by measuringthe uptake of [³H]thymidine. Media were removed, and cells wereincubated with 0.1 ml of trypsin until cells detached. Cells wereharvested onto 96-well filter plates using a sample harvester(FilterMate™ harvester; Packard Instrument Co., Meriden, Conn.). Theplates were then dried at 65° C. for 15 minutes, sealed after adding 40μl/well scintillation cocktail (Microscint™ 0; Packard Instrument Co.)and counted on a microplate scintillation counter (Topcount®; PackardInstrument Co.). Results, presented in Table 9, demonstrated that 80ng/ml zvegf4 had approximately 1.7-fold higher mitogenic activity onpericytes, 3.2-fold higher activity on aortic SMCs, and 2.6-fold higheractivity on aortic fibroblasts as compared to the buffer control.

TABLE 9 CPM Incorporated Pericytes HAoSMC AoAF Sample Mean St. dev. MeanSt. dev. Mean St. dev. Zvegf4, 80 ng/ml 96.7 18.2 488.7 29.6 177.0 1.0Zvegf4, 20 ng/ml 81.7 11.7 211.7 50.8 107.7 20.1 Zvegf4, 5 ng/ml 67.36.7 191.7 4.5 123.7 10.5 Buffer control 58.7 8.5 152.3 40.1 68.7 8.3

Example 13

Mice (C57BL6) were infected with a zvegf4-encoding adenovirus vector(AdZyvegf4) to determine the effects on serum chemistry, complete bloodcounts (CBC), body and organ weight changes, and histology. On day −1,the mice were tagged, individually weighed, and group normalized forseparation into treatment groups (4 mice per cage). Group 1 mice (n=8females, 7 males) received GFP (control) adenovirus, 1×10¹¹ particles.Group 2 mice (n=8 females, 6 males) received zvegf4 adenovirus, 1×10¹¹particles. Group 3 mice (n=8 females, 8 males) were untreated controls.On day 0, the mice received injections of the appropriate adenovirussolution. On day 10, blood was collected (under ether anesthesia) forCBCs and clinical chemistry measurements. On day 20, mice were weighedand sacrificed by cervical dislocation after collecting blood (underether anesthesia) for CBCs and clinical chemistry measurements. Tissueswere collected for histopathology. Observations were as follows:

-   Serum chemistry changes: AdZyvegf4 treated mice were hypoglycemic.    This effect increased in magnitude over time (day 10 vs. day 20).    Serum cholesterol levels were significantly increased (2-fold) at    both time points. Serum levels of albumin and the enzymes ALT, AST    and alkaline phosphatase were all significantly increased in    AdZyvegf4 treated mice. Serum calcium and total bilirubin were also    significantly increased, and became more elevated over time.-   CBC changes: AdZyvegf4-treated mice had significantly higher    lymphocyte count at both time points (mean >10). Platelet counts    were significantly lower at day 20. Red blood cell count was    significantly higher in females at day 10, significantly higher in    males at day 20.-   Body/organ weights: AdZyvegf4-treated males lost weight over the    course of the experiment. This result was significantly different    than control animals, which gained weight. There was no difference    among the female mice; all groups gained similar weight. Spleen    weight was significantly greater (approximately 4-fold) in all    AdZyvegf4-treated mice. Liver weight was also significantly greater    in all AdZyvegf4-treated mice. There was no significant difference    in kidney weight between groups.-   Histology: In the liver, proliferation of sinusoidal endothelial    cells was observed. In the spleen, proliferation of    reticuloendothelial cells was observed. In the kidney, proliferative    glomerulopathy was observed. While not wishing to be bound by    theory, this glomerulopathy may have been due to proliferation of    capillary endothelial cells. In the femurs, there was proliferation    of endosteal bone (mostly in trabecular bone), which in some cases    replaced most of the bone marrow. Proliferation of stromal cells was    also observed in bone. In the lung, there was increased frequency of    brochoaveolar lymphoid tissue.

Example 14

90 μg of recombinant zvegf4 protein was dissolved in 500 μl PBScontaining 2 mCi Na-125I (Amersham Corp.). One derivatized, nonporouspolystyrene bead (IODO-Beads®; Pierce, Rockford, Ill.) was added, andthe reaction mixture was incubated one minute on ice. The iodinatedprotein was separated from unincorporated ¹²⁵I by gel filtration usingan elution buffer of 10% acetic acid, 150 mM NaCl, and 0.25% gelatin.The active fraction contained 29 μg/ml ¹²⁵I-zvegf4 with a specificactivity of 3.0×10⁴ cpm/ng.

The following cell lines were plated into the wells of a 24-well tissueculture dish and cultured in growth medium for three days:

1. Human retinal pericytes, passage 6 (pericytes).

2. Rat stellate cells, passage 8.

3. Human umbilical vein endothelial cells, passage 4 (HUVEC).

4. Human aortic adventicial fibroblasts, passage 5 (AoAF).

5. Human aortic smooth muscle cells, passage 2 (AoSMC).

Cells were washed once with ice-cold binding buffer (HAM'S F-12containing 2.5 mg/ml BSA, 20 mM HEPES, pH 7.2), then 250 μl of thefollowing solutions was added to each of three wells of the culturedishes containing the test cells. Binding solutions were prepared in 5mL of binding buffer with 250 pM ¹²⁵I-zvegf4 and:

1. No addition.

2. 25 nM zvegf4.

3. 25 nM zvegf3.

4. 25 nM PDGF-AA.

5. 25 nM PDGF-BB

The reaction mixtures were incubated on ice for 2 hours, then washedthree times with one ml of ice-cold binding buffer. The bound¹²⁵I-zvegf4 was quantitated by gamma counting a Triton-X 100 extract ofthe cells.

Results, shown in Table 10, indicate binding of zvegf4 to pericytes,stellate cells, AoAF, and AoSMC, but not to HUVEC. The first columnrepresents total CPM ¹²⁵I-zvegf4 bound/well. The second column is¹²⁵I-zvegf4 bound/well when blocked with cold ligand. The differencebetween the two numbers represents specific binding.

TABLE 10 ¹²⁵I-zvegf4 Bound ¹²⁵I-zvegf4 Bound w/cold Cell Type (CPM)zvegf4 (CPM) 1. Pericytes 3083 +/− 864 623 +/− 60 2. Stellate Cells 2131+/− 450  413 +/− 164 3. HUVEC 485 +/− 91 227 +/− 13 4. AoAF 1544 +/− 131300 +/− 15 5. AoSMC 1628 +/− 203 440 +/− 46

Example 15

The zvegf4 gene was mapped to human chromosome 11 using the commerciallyavailable version of the Stanford G3 Radiation Hybrid Mapping Panel(Research Genetics, Inc., Huntsville, Ala.). This panel contains PCRableDNAs from each of 83 radiation hybrid clones of the whole human genome,plus two control DNAs (the RM donor and the A3 recipient). A publiclyavailable WWW server (http://shgc-www.stanford.edu) allows chromosomallocalization of markers. 20-μl reaction mixtures were set up in aPCRable 96-well microtiter plate (Stratagene, La Jolla, Calif.) and usedin a thermal cycler (RoboCycler® Gradient 96; Stratagene). Each of the85 PCR mixtures consisted of 2 μl buffer (10×KlenTaq PCR reactionbuffer, Clontech Laboratories, Inc., Palo Alto, Calif.), 1.6 μl dNTPsmix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1 μl sense primer,ZC22,685 (SEQ ID NO:37), 1 μl antisense primer, ZC22,686 (SEQ ID NO:38),2 μl of a density increasing agent and tracking dye (RediLoad, ResearchGenetics, Inc., Huntsville, Ala.), 0.4 μl of a commercially availableDNA polymerase/antibody mix (50× Advantage™ KlenTaq Polymerase Mix,obtained from Clontech Laboratories, Inc., Palo Alto, Calif.), 25 ng ofDNA from an individual hybrid clone or control, and x μl ddH2O for atotal volume of 20 μl. The reaction mixtures were overlaid with an equalamount of mineral oil and sealed. The PCR cycler conditions were aninitial 5-minute denaturation at 94° C.; 35 cycles of 45 secondsdenaturation at 94° C., 45 seconds annealing at 64° C. and 75 secondsextension at 72° C.; followed by a final extension for 7 minutes at 72°C. The reaction products were separated by electrophoresis on a 2%agarose gel. The results showed linkage of zvegf4 to the humanchromosome 11 framework marker SHGC-34226 with a LOD score of 14.90 andat a distance of 0 cR_(—)10000 from the marker. The use of surroundinggenes/markers positions Zvegf4 in the 11q22.3-q23.1 chromosomal region.

Example 16

The structure of recombinant zvegf4 was analyzed by Western blottingusing conventional techniques. Protein produced in the HaCaT humankeratinocyte cell line was electrophoresed under reducing andnon-reducing conditions, transferred to filters, and probed withantibodies to the interdomain and CUB domain regions of the protein.Reduced protein appeared as a single band having an apparent M_(r) ofapproximately 53 kD, consistent with a glycosylated, monomeric protein.Non-reduced protein appeared as a single band having an apparent M_(r)of approximately 85 kD, consistent with a disulfide-linked dimer.

Example 17

An expression plasmid containing full-length zvegf4 was constructed,using the expression vector pEZE2. pEZE2 is derived from pDC312 by theaddition of additional restriction enzyme recognition sites to themultiple cloning site. pDC312 and pEZE2 contain an EASE segment, asdescribed in WO 97/25420, which can improve expression of recombinantproteins two to eight fold in mammalian cells, preferably ChineseHamster Ovary (CHO) cells. The pEZE2 expression unit contains the CMVenhancer/promoter, the adenovirus tripartite leader sequence, a multiplecloning site for insertion of the coding region for the recombinantprotein, an encephalomyocarditis virus internal ribosome entry site, acoding segment for mouse dihydrofolate reductase, and the SV40transcription terminator. In addition, pEZE2 contains an E. coli originof replication and a bacterial beta-lactamase gene.

A zvegf4 DNA fragment was generated by PCR (ADVANTAGE2 PCR Kit,Clontech, Palo Alto, Calif.) with 5′ FseI and 3′ AscI sites for directcloning into the expression vector. The 5′ primer contained an FseIsite, Kozak sequence, and the first 21 basepairs of the native leadersequence for zvegf4 (ZC26,136; SEQ ID NO:43). The 3′ primer containedthe last 21 basepairs of zvegf4, a stop codon, and an AscI site(ZC26,137; SEQ ID NO:44). The PCR reaction included 1 μL of template(ESTEP plasmid zvegf4 perfl#3) and was run as follows: 94° C., 1 minute,1 cycle; then 25 cycles of 94° C., 30 seconds; 55° C., 30 seconds; 68°C., 1 minute; then a final extension cycle of 72° C. for 7 minutes.

The ESTEP plasmid zvegf4 perfl#3 contains the full-length human zvegf4fragment. This fragment was generated by PCR using 20 pm each ofZC22,341 (SEQ ID NO:45) and ZC22,342 (SEQ ID NO:46) primers and 3:L of athyroid library. The reaction was run as follows: 94° C., 1 minute, 1cycle; then 30 cycles of 94° C., 20 seconds; 66° C., 1.5 minutes; then afinal extension cycle of 72° C. for 5 minutes. The 1,272 bp product wasgel purified on a 1% TBE gel, and the DNA was extracted from the gelslab using the QIAQUICK Gel Extraction Kit (Qiagen, Valencia, Calif.).This 1,272 bp fragment was subcloned into pCR2.1 vector (Invitrogen,Carlsbad, Calif.), and designated zvegf4 perfl#3.

The PCR generated fragment was purified (QIAQUICK PCR clean-up kit,Qiagen, Valencia, Calif.) and digested with restriction enzymes AscI andFseI (New England Biolabs, Beverly, Mass.) in a single 100 μL reaction.Five micrograms of the expression vector pEZE2 were also digested withFseI and AscI in a single 100 μL reaction. The digested DNA wasfractionated by agarose gel electrophoresis and the DNA fragments wereisolated and purified (QIAQUICK Gel Extraction Kit, Qiagen).

Five microliters of the zvegf4 DNA fragment and 1 μL of the pEZE2 vectorfragment were ligated overnight at room temperature (New England BiolabsHigh Concentrated Ligase and supplied buffer). One microliter of theligation reaction was added to 25 μL of electrocompetant E. coli strainDH10B (Life Technologies) in a 0.2 cm cuvette. The mixture waselectroporated (BioRad E. coli Pulser) at 2.3 kv. To the cuvette, 1 mLof LB broth was added, and 100 μL of the mix was plated ontoLB/Ampicillin agar plates. The plates were incubated overnight at 37°C., and 8 isolated colonies were picked for DNA mini prep (QIAQUICKMini-Prep Kit, Qiagen). Individual clones were screened by PCR for thepresence of zvegf4 DNA, using the above-mentioned primers. DNAsequencing was performed on clones #1-6, to verify the correctfull-length sequence. One clone contained the correct expected sequenceand a Maxi prep of DNA was made (Qiagen Plasmid Maxi Kit, Qiagen).

CHO DG44 (Chasin et al., Som. Cell. Molec. Genet. 12:555-666, 1986) wereplated and allowed to grow to approximately 50% to 70% confluency overnight at 37° C. in MEM alpha media (JRH Biosciences, Lenexa, Kans.),7.5% fetal bovine serum (Hyclone, Logan, Utah), 1% L-glutamine (LifeTechnologies), 1% sodium pyruvate (Life Technologies), 1% HT solution(Life Technologies), and 1% Penicillin/Streptomycin (Life Technologies).The cells were then transfected with the plasmid pEZE2/zvegf4 byliposome-mediated transfection, using a 10:1 (w/w) liposome formulationof the polycationic lipid dioctaldecylamidoglycyl spermine, inserum-free (SF) medium formulation DMEM/F12—Life Technologies,Non-Essential Amino Acids-Life Technologies, 1% L-glutamine, 1% sodiumpyruvate. The plasmid pEZE2/zvegf4 was diluted in a final volume of 500μL of SF medium in a 15 mL conical tube, and 20 μL of TRANSFECTAM(Promega, Madison, Wis.) reagent was added, mixed well and incubated atroom temperature for 10 minutes. After incubation, 4.5 mL of SF mediumwas added to the DNA mixture and mixed well using a 5 mL pipette. Thecells were rinsed 3 times with SF medium, and the 5 mL of DNA solutionwas overlayed upon the cell monolayer. The cells were incubated at 37°C., 5% CO₂ for 2 hours. Then 6 mL of complete medium (MEM alpha, 7.5%FBS, 1% L-glutamine, 1% sodium pyruvate, 1% HT, 1% Pen/Strep) and thecells were incubated for a further 48 hours. After 48 hours, the cellswere trypsinized from the plate with 1 mL of 0.25% Trypsin/1 mM EDTA(Life Technologies) and quenched with 4 mL of complete medium withoutnucleosides (MEM alpha, 7.5% Dialysed FBS, 1% L-glutamine, 1% sodiumpyruvate, 1% Pen/Strep). Five hundred microliters of the cell suspensionwere transferred to plates containing 10 mL of complete medium withoutnucleosides. The cultures were grown for 14 days, until single coloniesthat were approximately 0.25 cm in diameter were present. Cloning rings(Bellco Glass, Inc., Vineland, N.J.) were used to isolate 24 singlecolonies, which were removed with trypsin, transferred to 6 well cellcluster plates (Costar, Corning, N.Y.), and incubated 4 days.

The cell wells were rinsed with SF medium and 2 mL of SF medium wasadded, and the culture was incubated for 24 hours. The conditioned SFmedium was concentrated approximately 20-fold using a 10K centrifugedevice (Millipore Corporation, Bedford, Mass.). Twenty-five microlitersof the concentrate was added to 15 μL of 4× Sample Buffer (Novex, SanDiego, Calif.) with 50 mM beta-Mercaptoethanol, and the mixture was runon a 4-12% NUPAGE gel (Novex). The proteins from the gel weretransferred to nitrocellulose membranes (Novex) and the blot was blockedwith 10% non-fat dry milk in Western A (0.25% gelatin, 50 mM Tris-HCl pH7.4, 150 mM NaCl, 5 mM EDTA, 0.05% Igepal CA-630) overnight at roomtemperature on a rotating shaker platform. The membrane was rinsed 3times in Western A. An antibody to the N-terminus of the zvegf4 proteinwas diluted at 1:3000 in 50 mL 5% non-fat milk in Western A. Theantibody solution was overlayed on the membrane and incubated at roomtemperature on a rocking platform for 1 hour. After the 1 hourincubation, the solution was discarded and the membrane rinsed 3 timeswith Western A and once with Western B (50 mM Tris pH 7.4, 5 mM EDTA,0.05% Igepal CA-630, 1 M NaCl, 0.25% Gelatin). The secondary antibody,an F(ab′)₂ fragment of Donkey-Anti-Rabbit-HRP (Amersham Corp., ArlingtonHeights, Ill.), was diluted in Western A at 1:3000, overlayed on themembrane, and incubated 1 hour at room temperature on a rockingplatform. The secondary antibody solution was discarded, and themembrane was washed 3 times in Western A and 3 times in Western B.Chemiluminescence was used to detect the full-length orprotease-digested N-terminus of zvegf4 according to the manufacturer'sinstructions (Pierce, Rockford, Ill.), and was analysed by LUMIANALYSER(Roche/Boehringer Mannheim, Mannheim, Germany). Four of the 12 cloneswere positive for zvegf4, and numbers 7 and 12 were trypsinized andtransferred to T175 flasks (Costar, Corning, N.Y.) in complete mediumwithout nucleosides.

Example 18

An expression construct encoding the growth factor domain of zvegf4 isprepared. A PCR fragment was generated (Clontech Advantage 2 PCR Kit)that contained a 5′ BamHI restriction site, an N-terminal EE tag, andzvegf4 amino acid residues 258-381 (stop codon included). The 5′ oligoprimer contains the BamHI site, an N-terminal EE tag sequence, andzvegf4 basepairs corresponding to the N-terminus of the growth factordomain (ZC27,116; SEQ ID NO:47). The 3′ oligo primer contains the last21 basepairs of zvegf4 (stop codon included) (ZC27,137; SEQ ID NO:48).The expression vector pZMP20 was used, which contains the CMV immediateearly promotor, a consensus intron from the variable region of mouseimmunoglobulin heavy chain locus, Kozak sequences, an optimized t-PAsecretory signal sequence (U.S. Pat. No. 5,641,655), multiplerestriction sites for insertion of coding sequences, a stop codon, and ahuman growth hormone terminator. The plasmid also contains an IRESelement from poliovirus, the extracellular domain of CD8 truncated atthe C-terminal end of the transmembrane domain, an E. coli origin ofreplication, a DHFR gene, the SV40 terminator, and the URA3 and CEN-ARSsequences required for replication in S. cerevisiae. The resultingplasmid is designated pZMP20/GFD.NEE. A 504 basepair fragment with a 5′FseI site and 3′ AscI site is isolated from this plasmid for ligationinto the pEZE2 expression vector (5′ FseI, 3′ AscI) for expression inCHO DG44 cells.

Example 19

A. Mouse Genomic Library Screen

A partial mouse zvegf4 sequence was obtained by probing a mouse genomiclibrary with a human zvegf4 restriction digest fragment containing theentire coding sequence. The probe was generated by digesting 8 μg of afull-length human zvegf4 plasmid with EcoR1 (Gibco BRL, Gaithersburg,Md.). The 1,289 bp fragment was gel purified on a 2.3% TBE gel and thecDNA was extracted from the agarose slab using the QIAQUICK GelExtraction Kit (Qiagen). The mouse genomic library was an embl3 SP6/T7lambda BamH1 cloned library (Clontech, Palo Alto, Calif.) plated on aK802 host lawn on 24 NZY plates, and represented 7.2×10⁵ pfus.

Twenty-four filter lifts were prehybridized in EXPRESSHYB solution(Clontech) containing 0.1 mg/ml salmon sperm DNA which had been boiled 5minutes, then iced. Hybridization took place overnight at 50° C. Sixtythree ng of the human fragment mentioned above were labeled with ³²Pusing the REDIPRIME II Random Prime Labeling System (Amersham Pharmacia,Buckinghamshire, England). Unincorporated radioactivity was removedusing a NucTrap push column (Stratagene, La Jolla, Calif.). Filters werehybridized in EXPRESSHYB solution containing 1.0×10⁶ cpm/ml zvegf4probe, 0.1 mg/ml salmon sperm DNA, and 0.5 μg/ml murine cot-1 DNA whichhad been boiled 5 minutes, then iced. Hybridization took place overnightat 50° C. Filter lifts were washed in 2×SSC, 0.1% SDS at roomtemperature for 2 hours, then the temperature was raised to 60° C. forone hour. Overnight exposure at −80° C. showed 7 putative primary hits.

A K802 host culture was prepared to plate the primary hits for asecondary screen. The 7 primary hits were picked with a Pasteur pipetand eluted in 1 ml SM (0.1 M NaCl, 50 mM Tris pH 7.5, mM MgSO₄, 0.02%gelatin) with a few drops of chloroform overnight at 4° C. After platingto determine titers, 10 times the number of plaques in the original pfuwere plated on NZY maxi plates with 10 mM MgSO₄/NZY top agarose and alawn of K802 cells for four of the primary hits and grown overnight at37° C. Lifts were done using HYBOND-N filters (Amersham Pharmacia). Thefilters were marked for orientation with a hot needle, denatured in 1.5M NaCl and 0.5 M NaOH for 10 minutes, then neutralized in 1.5 M NaCl and0.5 M Tris-HCl pH 7.2 for 10 minutes. The DNA was affixed to the filterusing a STRATALINKER UV crosslinker (Stratagene, La Jolla, Calif.) at1200 joules, and prewashed at 65° C. in prewash buffer consisting of0.25×SSC, 0.25% SDS and 1 mM EDTA, changing solution three times for atotal of 45 minutes to remove cell debris. Five lifts were put in eachvial, three vials total. Each vial of lifts was prehybridized overnightat 50° C. in 13 ml of EXPRESSHYB Hybridization Solution (Clontech) mixedwith 1.3 mg salmon sperm DNA which had been boiled 5 minutes, then iced.

Sixty-three ng of the human zvegf4 fragment was labeled for a probe asdescribed above. Each vial of filters was hybridized in 9 ml ofEXPRESSHYB Hybridization Solution mixed with 0.99 to 1.1×10⁶ humanzvegf4 probe, 0.5 μg/ml murine cot-1 DNA, and 0.9 mg/ml salmon sperm DNAwhich had been boiled 5 minutes, then iced. Hybridization took placeovernight at 50° C. Wash conditions described above for the primaryscreen were repeated for this secondary screen. Two of the 4 primaryputative hits that were tested came up positive after an overnightexposure at −80° C.

Isolated plaques #7c1 and #18b2 were eluted in 200 μl SM overnight at 4°C., and fresh host K802 cells were prepared. Serial dilutions rangingfrom 10² to 10³ were plated to obtain a titer estimate. Only #18b2 gaveany plaques (for a titer of 2.6 to 3.0×10³ phage per μl), and thisplaque was further pursued. Two plates with 10⁵ pfus per plate wereprepared for a phage DNA prep from plate lysates. Plates were grown at37° C. for 6 hours, until the phage were starting to get confluent, andthen 12 ml of SM per plate was added to elute the phage overnight at 4°C. At this point, plates were shaken at room temperature one hour, thesupernatant was removed, 1% chloroform was added, and supernatant wasshaken for 15 minutes. The DNA was prepped using the WIZARD Lambda PrepsDNA Purification System (Promega), sections IV and VI.

Plaque #18b2 DNA was cut with several restriction enzymes to generatefragments to run on a Southern gel. Digests were run on a 1% TBE agarosegel. The gel was soaked in 0.25 M HCl for 30 minutes, rinsed indistilled H₂O, soaked in 0.5 M NaOH and 1.5 M NaCl for 40 minutes withone solution change, and neutralized in 1.5 M NaCl and 0.5 M Tris-HCl(pH 7.2) for 40 minutes with one solution change. A TURBOBLOTTER RapidDownward Transfer System (Schleicher & Schuell, Keene, N.H.) was set upto transfer the DNA onto a Nytran/BA-S membrane (Schleicher & Schuell)overnight. The DNA was affixed to the Nytran using a STRATALINKER UVcrosslinker (Stratagene) at 1200 joules. The blot was prehybridizedovernight at 50° C. in 12 ml EXPRESSHYB Hybridization Solution(Clontech) mixed with 1.2 mg salmon sperm DNA which had been boiled 5minutes, then iced. Fifty nine ng of the human zvegf4 fragment waslabeled for a probe, as described above. Unincorporated radioactivitywas removed by chromatography using a commercially available push column(NUCTRAP column, Stratagene). Ten ml of EXPRESSHYB HybridizationSolution was mixed with 1.0×10⁶ cpm/ml of human zvegf4 probe, 0.5 μg/mlmurine cot-1 DNA, and 0.1 mg/ml salmon sperm DNA which had been boiled 5minutes, then iced, and then added to the blot. Hybridization took placeovernight at 50° C. The blot was washed as described above, and exposedto film overnight at −80° C.

The Southern gel had a fragment from the BamH1/Pst1 digest whichhybridized to the probe in the size range of 2.0 to 2.9 kb, which waspursued. Plaque 18b2 lambda DNA (2.8 μg) was cut with 20 units of BamH1(Boehringer Mannheim, Indianapolis, Ind.), and 20 μl Pst1 (LifeTechnologies) for 2 hours at 37° C. The digest was run on a 1% TBE gel,and a 2.0 kb doublet, as well as 2.7 kb/2.9 kb bands, were excised fromthe gel. The DNA was extracted from the agarose using the QIAQUICK GelExtraction Kit (Qiagen). The 18b2 fragments were ligated into aPBLUESCRIPTIIKS+vector (Stratagene) cut with BamH1, Pst1 and BamH1/Pst1.Three clones with a Pst1 insert, and 4 clones with a BamH1/Pst1 insert,from these ligations were digested with their respective insert siterestriction enzymes for another Southern blot to determine which was theoriginal hybridizing fragment. The 1% TBE gel was treated and the DNAwas transferred to the Nytran blot as described above. The blot wasprehybridized as above in 13 ml of hybridization solution. Fifty nine ngof the human zvegf4 fragment was labeled and unincorporatedradioactivity was removed as described above. Human zvegf4 probe(8.4×10⁵/ml cpm), 0.1 mg/ml of salmon sperm DNA, and 0.5 μg/ml of mousecot-1 DNA were boiled 5 minutes, iced 1 minute, and mixed with 7 ml ofEXPRESSHYB hybridization solution, then added to the blot. Hybridizationtook place overnight at 50° C. The same washing procedure was used asmentioned above. The blot was exposed to film for 3 hours at −80° C.,and both 2.0 kb band inserts strongly hybridized to the probe. Theseclones were sequenced and found to contain part of the murine zvegf4 cubdomain. Primers were designed from this sequence for a PCR cDNA screen.

B. PCR Screen of Mouse cDNA Panel

A panel of available in house and commercial mouse cDNAs were screenedwith 20 pm each of ZG26,317 (SEQ ID NO:49) and ZG26,318 (SEQ ID NO:50)primers. The PCR reaction conditions were as follows: 94° C., 2 minutes;then 35 cycles of 94° C., 10 seconds; 65° C., 20 seconds; 72° C., 30seconds; then ended with a 5 minute extension at 72° C. Embryo, salivarygland, neonatal skin and testis showed strong products of the predicted200 bp size.

C. Full Length Mouse zvegf4 Sequence

The in-house mouse testis arrayed library representing 9.6×10⁵ cloneswas screened by PCR using primers ZG26,317 (SEQ ID NO:49) and ZG26,318(SEQ ID NO:50) according to conditions specified above. This library wasdeconvoluted down to a positive pool of 250 clones. E. coli DH10B cells(Gibco BRL) were transformed with this pool by electroporation followingthe manufacturer's protocol. The transformed culture was titered andarrayed out to 96 wells at ˜20 cells/well. The cells were grown up inLB+amp overnight at 37° C. An aliquot of the cells was pelleted and PCRwas used to identify a positive pool. Thermocycler conditions were asdescribed above. The remaining cells from a positive pool were plated,and colonies were screened by PCR to identify a positive clone. Sequenceanalysis indicated that this clone, named “zvegf4 mpzp7x-6”, wasincomplete at the 5′ end and appeared to contain an intron at the 5′end.

The mouse salivary gland library representing 9.6×10⁵ clones was thenscreened by PCR using primers ZG26,317 (SEQ ID NO:49) and ZG26,318 (SEQID NO:50) according to conditions specified above. The library wasdeconvoluted down to a positive pool of 250 clones. This 250 clonal poolwas verified as having the 5′ end by RACE. Twenty pm each of ZG26,318(SEQ ID NO:50) and ZG14,063 (SEQ ID NO:51) primers and 3 μl of that poolwas used. The reaction was run as follows: 94° C., 2 minutes, then 5cycles of 94° C., 15 seconds; 70° C., 30 seconds; 30 cycles of 94° C.,15 seconds, 62° C., 20 seconds; 7° C., 30 seconds, and a final extensionat 72° C. for 7 minutes. The RACE product obtained upon sequencingconfirmed that this pool contained the initiation Met. The same protocolas described above was carried out to isolate a single clone from thepool. Sequence analysis revealed that this clone, named “zvegf4mpzp7x-7”, had a 225 bp deletion in coding compared to clone #6 (bp 865to by 1079 in the final sequence).

The sequences derived from zvegf4 mpzp7x-6 and from zvegf4 mpzp7x-7 werecombined to obtain a full-length mouse zvegf4 polynucleotide sequence(SEQ ID NO:52) and mouse zvegf4 polypeptide sequence (SEQ ID NO:53).

D. Full Length Mouse zvegf4 Clone

The full-length cDNA clone was generated by a two step ligation offragments from clone #6 and clone #7 from above. An EcoR1/Hind3 threeprime fragment was generated from clone #6 first. Nine μg of clone #6were digested with 15 units of EcoR1 (Gibco BRL, Gaithersburg, Md.) and15 units of Hind3 (Gibco BRL) for 2 hours at 37° C. The 528 bp fragmentwas gel purified on a 1% TBE gel, and the cDNA was extracted from thegel slab using the QIAQUICK Gel Extraction Kit (Qiagen). It was ligatedinto PBLUESCRIPTIIKS+(Stratagene) digested with EcoR1 and Hind3. Threeμg of a clone with this zvegf4 insert was digested with 15 units ofEcoR1 (Gibco BRL), gel purified on a 1% TBE gel, and the DNA wasextracted using the kit mentioned above. The 5′ EcoR1 zvegf4 fragmentfrom clone #7 was ligated into the EcoR1-digested clone mentioned above.This EcoR1 fragment was generated by digesting 8 μg of clone #7 with 30units of EcoR1 (Gibco BRL) for 2 hours at 37° C. The 754 bp fragment wasgel purified on a 1% TBE gel, and the DNA was extracted from the gelslab as mentioned above.

Example 20 A. Treatment of Naïve PC12 Cells with zvegf4 ConditionedMedium

HaCat cells were infected with a null adenovirus (zPar) as a control, orwith adenovirus expressing zvegf4. Conditioned medium (CM) from thesetransfected cells was assayed for its ability to induce neuriteoutgrowth in the PC12 Pheochromocytoma cell line (see Banker and Goslin,in Culturing Nerve Cells, chapter 6, “Culture and experimental use ofthe PC12 rat Pheochromocytoma cell line”; also, see Rydel and Greene, J.Neuroscience 7(11): 3639-53, November 1987).

Briefly, PC12 cell cultures (ATCC# CRL 1721) were propagated with RPMI1640 medium (Gibco/BRL, Gaithersburg, Md.), 10% horse serum (Sigma, St.Louis, Mo.), and 5% fetal bovine serum (FBS; Hyclone, Logan, Utah).Plastic culture dishes (Beckton Dickinson, Bedford, Mass.) were coatedwith rat tail collagen type I, and PC12 cells were plated into 24 wellplates at 2×10⁴ cells/ml in RPMI+1% FBS and incubated overnight at 37°C. in 5% CO₂. The PC12 culture medium was then removed, and replacedwith either zvegf4-CM or control-CM added in 2-fold dilutions (startingat 5× dilution). Recombinant human NGF (R+D, Minneapolis, Minn.) wasadded as a positive control at concentrations of 100 or 30 ng/ml. As anegative control, CM of the null adenovirus (zpar) was used. To test forsynergy of zvegf4 and NGF, additional wells of PC12 cells were treatedwith zvegf4-CM in combination with a suboptimal concentration of NGF (3ng/ml). The culture medium was replaced every second day with RPMI+1%FBS, until the total length of incubation reached 7 days.

The NGF-treated PC12 cells exhibited stable neurite outgrowth andneuronal differentiation. PC12 cells exposed to zvegf4-CM exhibitedmorphological changes, such as cell flattening and the appearance ofcells with short processes, suggesting differentiation into neuronallineage. For PC12 cells incubated with a suboptimal dose of NGF pluszvegf4-CM, an increase in a population of cells bearing short processeswas observed.

B. Treatment of Primed, Neurite-Bearing PC12 Cells with zvegf4Conditioned Medium

Zvegf4-CM and a control-CM (zpar) (as described in Example 20.A., above)were assayed for their ability to promote survival of differentiatedPC12 neurons (see Banker and Goslin, supra, Rydel and Greene, supra).

Briefly, PC12 cells were maintained as described in Example 20.A.,above, and were treated with appropriate doses of NGF to inducedifferentiation into cells that express the properties of post-mitoticsympathetic neurons. More specifically, PC12 cells were treated withrecombinant human NGF (R+D, Minneapolis, Minn.) at a concentration of 50ng/ml for 6 days, with a change of medium every other day. Cells wereplated into 24 well plates overnight, and the culture medium wasreplaced with zvegf4-CM or control-CM (in 2-fold dilutions, starting at5×), or with NGF as a positive control (starting with 100 ng/ml in3-fold dilutions).

Cultures were set up either with 1% FBS or serum-free culture (SF)medium. Cells were propagated over 9 days, with medium changes on everysecond day. Continuous treatment with NGF alone promoted the survival ofthe entire neuronal population and produced increasing neuriteoutgrowth. Zvegf4-CM promoted the survival of a subpopulation ofneurons, but did not induce additional neurite outgrowth. Cells culturedin control-CM degenerated.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method of stimulating the growth of bone tissue, the methodcomprising: applying to bone a growth-stimulating amount of a proteincomprising a first polypeptide disulfide-bonded to a second polypeptide,where each of said first and second polypeptides is from 113 to 138amino acid residues in length and comprises an amino acid sequencehaving at least 95% identity with amino acid residues 258-370 of SEQ IDNO:2.
 2. The method of claim 1, wherein each of said first and secondpolypeptide chains comprises an amino acid sequence having at least 95%identity with residues 250-370 of SEQ ID NO:2.
 3. The method of claim 1,wherein each of said first and second polypeptide chains comprisesresidues 250-370 of SEQ ID NO:2.
 4. The method of claim 1, wherein eachof said first and second polypeptide chains comprises an amino acidsequence having at least 95% identity with residues 246-370 of SEQ IDNO:2.
 5. The method of claim 1, wherein each of said first and secondpolypeptide chains comprises residues 246-370 of SEQ ID NO:2.
 6. Amethod of stimulating the growth of bone tissue, the method comprising:applying to bone a growth-stimulating amount of a protein produced bythe method comprising (a) culturing cells into which has been introducedan expression vector comprising the following operably linked elements:(i) a transcription promoter, (ii) a DNA segment encoding a polypeptidefrom 113 to 138 amino acid residues in length and comprising an aminoacid sequence having at least 95% identity with amino acid residues258-370 of SEQ ID NO:2, and (iii) a transcription terminator, wherebythe cell expresses the polypeptide encoded by the DNA segment; and (b)recovering the expressed protein.
 7. The method of claim 6, wherein theencoded polypeptide comprises an amino acid sequence having at least 95%identity with residues 250-370 of SEQ ID NO:2.
 8. The method of claim 6,wherein the encoded polypeptide comprises residues 250-370 of SEQ IDNO:2.
 9. The method of claim 6, wherein the encoded polypeptidecomprises an amino acid sequence having at least 95% identity withresidues 246-370 of SEQ ID NO:2.
 10. The method of claim 6, wherein theencoded polypeptide comprises residues 246-370 of SEQ ID NO:2.
 11. Amethod of stimulating the proliferation, differentiation, migration, ormetabolism of bone cells, the method comprising: exposing bone cells toan effective amount of a protein comprising a first polypeptidedisulfide-bonded to a second polypeptide, where each of said first andsecond polypeptides is from 113 to 138 amino acid residues in length andcomprises an amino acid sequence having at least 95% identity with aminoacid residues 258-370 of SEQ ID NO:2.
 12. The method of claim 11,wherein each of said first and second polypeptide chains comprises anamino acid sequence having at least 95% identity with residues 250-370of SEQ ID NO:2.
 13. The method of claim 11, wherein each of said firstand second polypeptide chains comprises residues 250-370 of SEQ ID NO:2.14. The method of claim 11, wherein each of said first and secondpolypeptide chains comprises an amino acid sequence having at least 95%identity with residues 246-370 of SEQ ID NO:2.
 15. The method of claim11, wherein each of said first and second polypeptide chains comprisesresidues 246-370 of SEQ ID NO:2.
 16. A method of stimulating theproliferation, differentiation, migration, or metabolism of bone cells,the method comprising: exposing bone cells to an effective amount of aprotein produced by the method comprising (a) culturing cell into whichhas been introduced an expression vector comprising the followingoperably linked elements: (i) a transcription promoter, (ii) a DNAsegment encoding a polypeptide from 113 to 138 amino acid residues inlength and comprising an amino acid sequence having at least 95%identity with amino acid residues 258-370 of SEQ ID NO:2, and (iii) atranscription terminator, whereby the cell expresses the polypeptideencoded by the DNA segment; and (b) recovering the expressed protein.17. The method of claim 16, wherein the encoded polypeptide comprises anamino acid sequence having at least 95% identity with residues 250-370of SEQ ID NO:2.
 18. The method of claim 16, wherein the encodedpolypeptide comprises residues 250-370 of SEQ ID NO:2.
 19. The method ofclaim 16, wherein the encoded polypeptide comprises an amino acidsequence having at least 95% identity with residues 246-370 of SEQ IDNO:2.
 20. The method of claim 16, wherein the encoded polypeptidecomprises residues 246-370 of SEQ ID NO:2.